EP2125052A2 - Composition thérapeutique et utilisation d'une substance acellulaire - Google Patents

Composition thérapeutique et utilisation d'une substance acellulaire

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Publication number
EP2125052A2
EP2125052A2 EP08706849A EP08706849A EP2125052A2 EP 2125052 A2 EP2125052 A2 EP 2125052A2 EP 08706849 A EP08706849 A EP 08706849A EP 08706849 A EP08706849 A EP 08706849A EP 2125052 A2 EP2125052 A2 EP 2125052A2
Authority
EP
European Patent Office
Prior art keywords
cell
cells
therapeutic composition
bone
free
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Withdrawn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
EP08706849A
Other languages
German (de)
English (en)
Inventor
Michael Thie
ÖZER Degistirici
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.)
Stiftung Caesar Center of Advanced European Studies and Research
Original Assignee
Stiftung Caesar Center of Advanced European Studies and Research
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 Stiftung Caesar Center of Advanced European Studies and Research filed Critical Stiftung Caesar Center of Advanced European Studies and Research
Publication of EP2125052A2 publication Critical patent/EP2125052A2/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Withdrawn legal-status Critical Current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
    • A61L27/36—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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3612—Cartilage, synovial fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the invention relates to a therapeutic composition, a method for producing a therapeutic composition and the use of a cell-free substance, in particular a cell-free bone or cartilage matrix.
  • stem cells From stem cells, additional stem cells can be created by division and / or differentiated cells emerge, ie stem cells are able to divide asymmetrically.
  • a stem cell retains its ability to divide over a very long period, often even throughout the life of the organism. Triggered by specific signals during the development of the organism, a stem cell can differentiate into different cell types, which then form the organism. A distinction is generally made between embryonic and adult stem cells.
  • Embryonic stem cells obtained from an embryo up to the 8-cell stage are termed totipotent. From these cells develops the complete organism (human). Embryonic stem cells from later stages of blastocysts are said to be pluripotent, since they can generally be used to differentiate all types of endoderm body cells (wall cells of the digestive tract), mesoderms (muscles, bones, blood cells), and ectoderms (skin cells and nerve tissue). but no complete organism anymore. However, for ethical reasons and because of problems with the molecular control of cell differentiation, ES cells have not yet been therapeutically applicable.
  • a cells are undifferentiated cells that are located in a differentiated tissue and that produce specialized cells that correspond to those of the differentiated tissue.
  • ES cells can also differentiate into cell types that are associated with another tissue.
  • Adult stem cells that are found in organs, in the bone marrow or even in the umbilical cord can no longer differentiate as freely as embryonic stem cells.
  • adult stem cells do not have the same potential for differentiation as embryonic stem cells, they nevertheless have potential for ciliary leaf differentiation. One calls it therefore as multipotent.
  • mesenchymal stem cells derived from the mesoderm may also differentiate into neuronal cells that otherwise develop from the ectoderm.
  • AS cells are able to differentiate into a cell type that does not correspond to that of their parent tissue after relocation to another tissue type.
  • Adult stem cells are available in every organ, for example in the bone marrow. However, the removal of bone marrow is a complicated and risky surgical technique.
  • the production of stem cells from tooth tissue is a less expensive alternative, as described for AS cells from dental follicles in WO 03/066840 A2.
  • Such stem cells from readily available tissue, for example, open the perspective of tissue replacement by the body's own cells.
  • the tendency for malignant degeneration in implantation of adult stem cells seems to be smaller than in embryonic stem cells.
  • Adult stem cells are therefore of increasing importance for the development of innovative therapeutic approaches.
  • EP 0 957 944 B1 discloses a method for producing a bone matrix populated with osteogenic cells which, after implantation in vivo, is intended to stimulate new bone formation by inducing differentiation processes in the cells of the surrounding tissue.
  • first osteogenic progenitor cells from explants of somatic cells of the skin, cartilage, bone or the dental apparatus, in particular human stem cells from the iliac crest, isolated.
  • the isolated cells are stimulated in vitro by the addition of growth factors, such as Bone Morpho-genetic Proteins (BMPs), thereby stimulating the synthesis of bone matrix.
  • BMPs Bone Morpho-genetic Proteins
  • the bone matrix can then be separated from the cells by enzymatic treatment or freezing and subsequent washing to form a cell-free bone matrix.
  • This cell-free bone matrix is then repopulated with fresh osteogenic cells, creating an autologous cell-loaded bone substitute.
  • osteogenic cells and bone matrix are prepared separately and then brought together again to produce a bone substitute material.
  • the use of a cellular bone substitute has the disadvantage that this due to the antigenic properties of the living cells can lead to immunogenic rejection reactions, if the cells are not derived from the person to be treated. Such a cell-loaded bone matrix is therefore not universally applicable in therapy.
  • a therapeutic composition which consists of at least one cell-free substance, which has emerged from stimulated stem and / or progenitor cells. Because the therapeutic composition is cell-free and does not contain any typically antigenic cell components, immunogenic reactions do not occur in vivo in the therapeutic application. Independently of the origin of the parent and / or precursor cells, the composition according to the invention can therefore be used universally for therapy and use its natural regenerative potency for tissue replacement, for example a suitable bone and / or cartilage assembly, in a highly efficient manner.
  • the therapeutic composition of the invention can stimulate the regeneration of destroyed tissue in vitro and in vivo, although it itself no longer contains stem cells.
  • the substances contained in the stimulated stem and / or progenitor cells or produced by these cells are sufficient to enable or at least support effective tissue replacement therapy.
  • the composition of the invention contains no antigenic cell components and can thus be used in all patients without the risk of immunogenic reactions. This allows industrial production of the preparation and thus ensures constant availability.
  • the cell-free substance is a cell culture in which the living cells are killed and / or cell components are at least partially removed.
  • all antigenic components such as cell surface antigens or other proteins, must be effectively removed.
  • Nucleic acids should also be removed or at least completely degraded to eliminate any risk of introducing genetic material.
  • the cell-free substance comprises cell extract and / or cell secretions, d. H. antigen-free cell components, cell contents and / or substances produced by the stimulated cells.
  • the cell-free substance is a developing bone matrix.
  • the formation of this matrix takes place on the basis of ectomesenchymal progenitor cells and is similar in its essence to that of embryonic bone formation, so that the composition according to the invention can be used or used with regard to growth-promoting modulators in bone replacement therapy (catalyst in bone formation).
  • the cell-free bone matrix allows a targeted stimulation of bone formation in vivo, without the possibility of immunogenic reactions.
  • patients who due to deficiencies in bone structure, or only with difficulty with implants, eg. As dental implants can be prepared by introducing the therapeutic composition into the defective tissue in a simple manner and without risk.
  • the cell-free substance can also be cartilage matrix, so that defective cartilage tissue can also be regenerated by means of the composition according to the invention.
  • the therapeutic composition can also be used to treat injuries or degenerative diseases in joints.
  • stem and / or progenitor cells that form the cell-free substance in an advantageous embodiment of the invention multipotent cells from a
  • stem cells of follicle or pulp may be used.
  • stem cells from a cushion-like soft tissue which is located directly on the apical side of a extracted immature tooth below the papilla is localized.
  • the cushion-like soft tissue is advantageously a cell composite consisting of a plant of an impacted and / or retained tooth in the developmental phase between the occurrence of the bony alveolar fundus and the completion of the rooting.
  • the cushion-like soft tissue should be separated from the tooth .
  • the tissue selected in this way makes it possible to isolate ectomesenchymal stem or progenitor cells which, because of their multipotency, can be differentiated into different cell types, for example bone or cartilage cells.
  • the choice of the correct tissue of origin for the isolation of the stem cells by the method according to the invention is an essential factor for a high yield of multipotent stem cells.
  • the isolated cells are ektomesenchymale stem and / or progenitor cells, which can be stimulated osteogenically and / or chondrogenically after isolation from the tissue composite.
  • the cell-free substance has a defined ratio of calcium to phosphate.
  • a defined Calcuim / Phopshat ratio for example, bone matrix can be produced, which corresponds to a young bone or an old bone.
  • the cell-free substance according to the invention can thus be tailored specifically for the desired application.
  • the calcium / phosphate ratio provides evidence of the age of the bone (Dorozhkin, SV, Epple, M. 2002).
  • a therapeutic composition comprising a cell-free substance having a defined calcium / phosphate ratio, preferably a calcium / phosphate ratio of between 0.5 and 2.0 so as to provide a curative optimized for the desired therapy, which has defined properties.
  • the cell-free substance can also be a defined The ratio of calcium phosphate to collagen, so that the quality of the therapeutic composition can be precisely adjusted.
  • the object is further achieved according to the invention by a process for preparing a therapeutic composition in which stem and / or progenitor cells are isolated, cultured in vitro and stimulated for subsequent differentiation, wherein the cells are killed after differentiation to produce a cell-free substance and / or at least partially removed, and wherein the cell-free substance is prepared for therapeutic use.
  • a cell-free substance which has emerged or was produced from stimulated stem and / or progenitor cells, is directly brought into a therapeutic application form, without colonizing them again with stem cells.
  • the therapeutic composition prepared according to the method of the invention can stimulate the regeneration of destroyed tissue in vitro and in vivo, although it is not populated with fresh stem cells. It appears that the substances contained in the stimulated stem and / or progenitor cells or produced by these cells are sufficient to enable or at least support effective tissue replacement therapy. The production process is thereby considerably simplified and moreover cost-effective, since parallel to the production of the cell-free substance no stem or progenitor cells have to be cultivated any more.
  • the cell-free substance is preferably comminuted, pulverized and / or freeze-dried, so that an easily handled and processed product is formed.
  • the cell-free substance can then be prepared according to the invention, for example in the form of a paste, ointment or suspension.
  • the cell-free substance or the therapeutic composition can be easily introduced into the tissue to be treated. This gentle type of application is derem advantage, as this additional, the patents incriminating interventions can be avoided.
  • the stem and / or progenitor cells osteogenically and / or chondrogenically.
  • the stem and / or progenitor cells can be stimulated in such a way that they form or produce a bone or cartilage matrix.
  • This matrix and any other substance which is produced in vitro without additional aids such as, for example, carrier materials can then be freed by enzymatic, chemical or mechanical treatment of living cells and / or antigenic cell constituents and subsequently used to prepare the composition according to the invention.
  • Living cells should at least be killed and / or antigenic cell components should be removed as completely as possible. Nucleic acids should also be removed or at least completely degraded to eliminate any risk of introducing genetic material.
  • the intact or minced bone mass is treated with a hypotonic solution (eg, deionized water) and / or treated in liquid nitrogen for ice nucleation to destroy the cell membranes and lyse the cells.
  • a hypotonic solution eg, deionized water
  • the samples are washed out with shaking in saline solution (eg 3M NaCl) and / or acid / base (eg 0.15-1% per acidic acid PAA), and / or extracted in Triton-X100 (0.1% -1%) and / or SDS (0.1% -1%) for different times.
  • saline solution eg 3M NaCl
  • acid / base eg 0.15-1% per acidic acid PAA
  • Triton-X100 0.1% -1%)
  • SDS 0.1% -1%)
  • RNA and DNA released nucleic acids
  • the bone matrix is treated with RNAse and DNAse.
  • the cell-free bone matrix substance is washed with buffer solution (eg PBS), dialyzed and lyophilized, pulverized and prepared in the form of a paste, ointment or suspension.
  • buffer solution eg PBS
  • the duration of the stimulation for adjusting certain properties of the cell-free substance can be varied.
  • a defined ratio of calcium to phosphate and / or of calcium phosphate to collagen can be set, for example, by the choice of a specific stimulation duration.
  • the duration of stimulation ie the duration of the cultivation of the stem cells in the presence of the stimulating substances, is according to the invention between 15 and 50 days, preferably between 20 and 45 days, particularly preferably between 21 and 42 days.
  • the degree of maturation and the quality of the cell-free substance can be influenced and thus set in a defined manner.
  • the shorter the stimulation time the lower the degree of maturity or mineralization (small calcium / phosphate ratio) of the generated bone matrix or cell-free substance.
  • the longer the stimulation time the higher the degree of maturity or mineralization (large calcium / phosphate ratio) of the generated bone matrix or cell-free substance.
  • the method according to the invention thus makes it possible in a particularly advantageous manner to selectively adjust the properties of the process product by selecting a specific stimulation duration.
  • the stem cells are isolated from a soft tissue of a tooth, such as, for example, follicles or pulp.
  • the stem cells are isolated from a pillow-like soft tissue that is directly localizable on the apical side of an extracted immature tooth underneath the papilla.
  • the cushion-like soft tissue results from an implantation of an impacted and / or retained tooth in the development phase between the occurrence of the bony alveolar fundus and the Conclusion of rooting is won.
  • the cushion-like soft tissue In order to be able to isolate the desired multipotent stem cells, after the surgical removal of the tooth, the cushion-like soft tissue should be located along a macroscopically visible boundary between the tooth pincushion soft tissue and the papilla, preferably below an imaginary line between the developing root processes, are separated from the tooth.
  • the tissue selected in this way allows the isolation of ectomesenchymal stem or precursor cells, which can be differentiated into different cell types, for example bone or cartilage cells, because of their multipotency.
  • the choice of the correct tissue of origin for the isolation of the stem cells by the method according to the invention is an essential factor for a high yield of multipotent stem cells.
  • the tissue composite can be dissolved for further cultivation of the cells by enzymatic treatment, preferably with collagenase / dispase.
  • the cells can also be separated after the extraction from the tissue composite.
  • the cells isolated by the method according to the invention are ectomesenchymal stem and / or progenitor cells, which can be stimulated osteogenically and / or chondrogenically in vitro after isolation from the tissue composite.
  • Each therapeutic composition prepared by the method according to the invention is also an object of the invention.
  • the cell-free substance described above can, as already explained, be used for therapeutic purposes in the context of a tissue replacement therapy.
  • the invention further encompasses the use of a cell-free cell extract and / or cell secretion for therapeutic purposes in tissue replacement therapy and the use of a cell-free bone or cartilage matrix for therapeutic purposes in tissue replacement therapy.
  • FIG. 1 (a) is a photograph of an extracted wisdom tooth with apical soft tissue (apical pillow) and (b) a micrograph of cells isolated from the apical pillow;
  • FIG. 2 is a scanning electron micrograph of bone formations after 28 days of incubation of the isolated cells after glutaraldehyde fixation (coated with gold), (a) control, (b) external view and (c) sectional view,
  • Figure 1 shows a surgically removed wisdom tooth with apical pad (a).
  • the apical pad is separated from the tooth along the macroscopically visible boundary between the soft tissue of the pillow and the papilla below the imaginary line between the tooth roots, washed several times with PBS (sterile), and then minced with a scalpel.
  • the tissue thus obtained is disrupted with collagenase / dispase.
  • the cultures are cultured at 37 ° C. in DMEM LG 10% FCS.
  • the isolated cells grow as fibroblastoid cells in cultures (b).
  • FIG. 2 shows scanning electron micrographs of bone formations after 28 days of incubation of the isolated cells.
  • the cells are cultured in control medium (a) and in osteogenic stimulating medium (b).
  • the cells cultured in osteogenic stimulating medium form a bone-like formation (b) that organizes bone-like organized from the inside is (c) while the cells treated with control medium alone do not form bone formation and grow as an adherent dressing (a).
  • FIG. 3 shows microscopic images of osteogenic stimulated cultures.
  • the osteogenically stimulated cultures are stopped after 42 days and fixed with 4% PFA. Subsequently, the formed bone formations are embedded in paraffin and then cut.
  • the sections are immunohistochemically stained with a first antibody against osteocalcin or collagen type I (both are bone markers). The bound antibodies are detected with the DAB kit according to the manufacturer's instructions (R & D). To detect calcification, sections are stained with alizarin red (1.2% alizarin red to pH4.2). The strongly red colored areas are calcified or bone-like areas.
  • Dental stem cells have a specific pattern of active substances that control the structure of different types of tissue.
  • a specially isolated subpopulation of human dental stem cells in a cell biological process is stimulated to develop a tissue similar to the young bone.
  • the resulting highly specific matrix is then processed into a non-cellular product that significantly accelerates the bone formation and thus the course of the healing phase and the ingrowth behavior of implants.
  • the special property of this matrix is used to stimulate the complex bone structure physiologically correct in a wound due to their composition.
  • stem / progenitor cells are osteogenically stimulated under suitable conditions to give rise to a bone-like tissue bandage.
  • the osteogenic stimulation can be carried out, for example, by incubation or cultivation of the stem cells with 10 -7 M dexamethasone, 50 ⁇ g / ml ascorbic acid 2-phosphate and 10 mM ⁇ -glycerol phosphate. make bones comparable.
  • the matrix is processed and processed into a product. The entire process is purely biological and does not require any scaffold.
  • the starting material is a differentiated bone tissue, which can only be formed from young dental stem cells, the so-called dNC-PCs, which are osteogenically stimulated, in a self-generating culture system.
  • the culture system is particularly advantageous because the three-dimensional bone tissue together with its specifically formed intercellular substances is organized exclusively by dNC PCs themselves and in its structure and function is subject exclusively to its own formative and ordering principles.
  • the bone tissue is thus developing, immature bone tissue, which is of special construction in its structure and strength, its degree of mineralization and its composition of basic substance components.
  • This bone tissue is processed into a non-cellular product (basic bone substance), which accelerates the bone formation and thus the course of the healing phase and the ingrowth of implants (scaffolds) in the patient.
  • This bone tissue can be harvested at different levels of ripeness, following phases of matrix and mineralization, and processed in ripeness-dependent processes.
  • the maturity levels are determined by the differentiation states of the dNC PCs or the structuring and composition of the matrix
  • the removal of the cells and the production of a cell-free substance is carried out by physical, chemical and biological methods.
  • a hypotonic solution eg deionized water
  • the intact or crushed bone mass is treated with a hypotonic solution (eg deionized water) and / or treated in liquid nitrogen for ice crystal formation.
  • the samples are washed out with shaking in saline solution (eg 3M NaCl) and / or acid / base (eg 0.15-1% per acetic acid PAA), and / or Triton X100 (0.1% -1%) and / or SDS (0.1% -1%) extracted for different times.
  • the cell-free substance is thus based in principle on the synthesis performance of a neural crest cell.
  • We isolate these cells from the cells of a cushion-like soft tissue, which is located immediately on the apical side of an extracted immature tooth below the papilla (where they have settled in their niche) and describe their properties as multipotent dNC PCs ( dental neural crest-derived progenitor cells).
  • dNC PCs dental neural crest-derived progenitor cells.
  • This aggregate which is completely self-contained, basically consists of a "core” and a "shell".
  • the nucleus is formed by inner cells, the shell by outer cells.
  • Both cell types can be distinguished phenotypically, but are initially derived from dNC PCs as a common precursor.
  • the core of the aggregate consists of vital cells, all of which are related to each other and belong to the family of osteoblasts and osteoblast helper cells.
  • the shell of the aggregate consists of vital cells.
  • the inner cells begin to separate an embryonic matrix with increasing culture duration, which is initially not mineralized. Only at long culture times, there is a successive storage of mineral (variants of Ca phosphates up to the hydroxylappatite) and thus the formation of hard substance.
  • the process of mineralization occurs only inside the aggregate and is likely bound to the presence of the shell. Mineralization is an active process, ie it is dependent from the inner cells and the specific milieu of the nucleus.
  • the interior of the aggregate is the starting product for the cell-free substance according to the invention, which can be harvested at different degrees of ripeness. For harvest, the aggregate is broken up.
  • the outer cells are removed by enzyme treatment or Immunosurgery. What remains is the core of the unit, which is not used directly but is still being processed.
  • the cells are isolated with enzyme and detergent. What remains can be separated into an organic and an inorganic phase. Each phase can be further modified - the organic with enzyme, the inorganic with acid and / or base. Depending on the degree of maturity of the unit and depending on the process of preparation results in each case a different product.

Abstract

L'invention concerne une composition thérapeutique, un procédé de production d'une composition thérapeutique et l'utilisation d'une substance acellulaire, en particulier d'une matrice osseuse ou cartilagineuse acellulaire. La composition thérapeutique selon l'invention est constituée d'au moins une substance acellulaire provenant de cellules souches et/ou précurseurs stimulées. Du fait que cette composition thérapeutique est acellulaire et ne contient aucun constituant cellulaire antigène, aucune réaction immunogène n'est provoquée lors de l'application thérapeutique in vivo. La composition selon l'invention peut être utilisée en thérapie de façon universelle indépendamment de l'origine des cellules souches et/ou précurseurs et la capacité régénérative naturelle desdites cellules peut être exploitée de manière très efficace pour le remplacement de tissus, par exemple pour une construction osseuse et/ou cartilagineuse appropriée.
EP08706849A 2007-02-01 2008-02-01 Composition thérapeutique et utilisation d'une substance acellulaire Withdrawn EP2125052A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007005946A DE102007005946A1 (de) 2007-02-01 2007-02-01 Therapeutische Zusammensetzung und Verwendung einer zellfreien Substanz
PCT/DE2008/000184 WO2008092440A2 (fr) 2007-02-01 2008-02-01 Composition thérapeutique et utilisation d'une substance acellulaire

Publications (1)

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EP2125052A2 true EP2125052A2 (fr) 2009-12-02

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US (1) US20100104641A1 (fr)
EP (1) EP2125052A2 (fr)
DE (1) DE102007005946A1 (fr)
WO (1) WO2008092440A2 (fr)

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JP6929346B2 (ja) * 2016-07-04 2021-09-01 カンステム バイオテック カンパニー リミテッド 軟骨無細胞破砕物及び幹細胞を含む軟骨分化促進用複合体及びその用途
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US20100104641A1 (en) 2010-04-29
DE102007005946A1 (de) 2008-08-14

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