WO2009003375A1 - Tendon d'ingénierie tissulaire et procédés de construction in vitro de celui-ci - Google Patents

Tendon d'ingénierie tissulaire et procédés de construction in vitro de celui-ci Download PDF

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WO2009003375A1
WO2009003375A1 PCT/CN2008/070566 CN2008070566W WO2009003375A1 WO 2009003375 A1 WO2009003375 A1 WO 2009003375A1 CN 2008070566 W CN2008070566 W CN 2008070566W WO 2009003375 A1 WO2009003375 A1 WO 2009003375A1
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Prior art keywords
cells
tendon
seed
fibroblasts
adipose
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PCT/CN2008/070566
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English (en)
French (fr)
Inventor
Yilin Cao
Wei Liu
Feng Xu
Dan Deng
Hong Li
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Shanghai Tissue Engineering Life Science Co. Ltd.
Shanghai 9th People's Hospital, Shanghai Jiaotong University School of Medicine
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Application filed by Shanghai Tissue Engineering Life Science Co. Ltd., Shanghai 9th People's Hospital, Shanghai Jiaotong University School of Medicine filed Critical Shanghai Tissue Engineering Life Science Co. Ltd.
Priority to JP2010513625A priority Critical patent/JP2010531683A/ja
Priority to EP08715302A priority patent/EP2172231A4/en
Publication of WO2009003375A1 publication Critical patent/WO2009003375A1/zh
Priority to US12/648,827 priority patent/US20100197020A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—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
    • A61L27/3839—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 the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/386—Ligaments, tendons
    • 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
    • A61L27/3886—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 comprising two or more cell types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/066—Tenocytes; Tendons, Ligaments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/08—Muscles; Tendons; Ligaments
    • 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
    • A61L2430/10—Materials or treatment for tissue regeneration for reconstruction of tendons or ligaments

Definitions

  • This invention relates to the field of medical and biomedical engineering, and more particularly to a method and use for the preparation of tissue engineered tendons in vitro using dermal fibroblasts and/or adipose derived cells. Background technique
  • the tendon is a bundle of dense collagen fibers that connect the skeletal muscle to the bone and is part of the skeletal muscle.
  • the structure of the tendon can be seen as a continuation and degeneration of the muscle.
  • the fresh specimens are silvery white, cord-like, and tough.
  • the surface is corrugated. If the pulling exceeds 4% of the relaxed state, the corrugation disappears, and the ripple is reproduced after the tension is canceled.
  • collagen fibers enter the endomysium and attach to the sarcolemma of the muscle fibers at the iliac-bone junction.
  • the tendon bundle is directly continuous to the bone and periosteum, and most of the collagen fibers enter the bone, forming Sharpey wear. fiber.
  • the tendon transmits the force generated by the contraction of the muscles, pulling the bones and causing them to move.
  • the tendon itself does not have the ability to contract, but it can resist a lot of tension.
  • Tendon defect is a problem often encountered in clinical work.
  • the treatment methods for tendon defect are: 1. Autologous tendon transplantation. 2. Allogeneic tendon transplantation. 3. Replacement of tendon grafts.
  • the artificial tendon substitute has a good biomechanical strength in the near future, it will eventually degrade in the long term, and it may cause inflammation, fibrosis and tendon adhesion, and even substitute excretion.
  • the implanted scaffold is a material that has not been degraded, and its decomposition products are mostly acidic, which can directly lead to inflammatory reaction, scar formation and tendon adhesion, which directly affects the repair effect of tendon.
  • the mechanical properties of the cell-material complex are greatly reduced with the degradation of the material, and the tendon defect at the high tension site cannot be repaired. Therefore, although there are more in vivo experiments in the construction and repair of tendons, due to the above problems, there is still a considerable distance from clinical application.
  • tissue engineering techniques to construct tendon tissue, repair tendon tissue defects, and achieve industrial production is to obtain a large number of normal functioning tendon cells.
  • the tendon cells lose the ability to secrete the matrix after multiple passages, and it is difficult to obtain a large number of functionally active tendon cells to repair large defect tissues.
  • the second key is to build a relatively mature tendon tissue with certain mechanical properties and most of the material has been degraded. Therefore, exploring a wider range of seed cell sources and more optimized in vitro construction techniques has become the focus of tissue engineering tendon research.
  • MSCs bone marrow stromal cells
  • dermal fibroblasts dermal Fibrob last DF
  • MSCs are a group of cells with multipotential differentiation potential, which can differentiate into a variety of mesenchymal tissue cells under specific induction conditions.
  • Awad et al. isolated and cultured MSCs and complexed with collagen to form a cellular collagen complex implanted into the artificial defect patellar ligament.
  • control group collectively applied collagen
  • Growth factors are a class of polypeptide factors that affect cellular activity through intercellular signaling, which promote or inhibit cell proliferation in vivo or in vitro.
  • Many scholars directly use growth factors to study and confirm their promotion of tendon injury repair in vitro and in vivo.
  • people use genetically modified technology to transfer genetic information to cells to alter cell function. Using this technology, target cells increase or inhibit the synthesis and secretion of proteins (such as growth factors), regulate cell growth, and participate in tissue repair processes. Successful application of this method allows cells and tissues to synthesize secreted cytokines to regulate their growth as required.
  • Some scholars have successfully transferred genes into tendons by different methods. The LacZ marker gene has been continuously expressed for 6 weeks after application to the patellar tendon.
  • Lou et al. used transgenic technology to transfer the LacZ marker gene into the tendon.
  • the gene can be detected after 75 days, and the adhesion shape after reducing and preventing the tendon injury is proposed.
  • the present invention is directed to a tissue engineered human tendon graft.
  • tissue engineered tendon graft comprising:
  • seed cells said seed cells being seeded with said biodegradable material, and seed cells selected from the group consisting of: (i) fibroblasts, (ii) adipose-derived cells, or (iii) 1 : 10000 — 10000 : 1 mixture of fibroblasts and adipose derived cells.
  • the tissue engineered tendon graft has a maximum tension of 10 - 80N.
  • the seed cell is a mixture of fibroblasts or fibroblasts and adipose derived cells.
  • the seed cell is present in an amount of 1 X 10 5 cells/ml-5 X 10 8 cells.
  • the biodegradable material is in the form of a cord.
  • the fibroblasts and adipose derived cells are derived from autologous or allogeneic.
  • the pharmaceutically acceptable biodegradable material is selected from the group consisting of polylactic acid, polyglycolic acid, polyhydroxybutyric acid, polyanhydride, polyazo phosphate, polyamino acid, pseudopolyamino acid Polyortic acid Ester, polyester urethane, polycarbonate, polyethylene glycol, polyethylene oxide, polydioxanone, collagen, gelatin, glycosaminoglycan, chitosan, chitin, alginate, Calcium alginate gel, acellular matrix, and mixtures of various types and ratios.
  • a method of preparing a tissue engineered tendon graft as described above comprising the steps of:
  • the seed cells are mixed with a pharmaceutically acceptable biodegradable material to obtain a seed cell-biomaterial complex, wherein the seed cells are seeded with the biodegradable material, and the seed cells are selected from the group consisting of: a mixture of fibroblasts, (ii) adipose-derived cells, or (iii) 1:10000 ⁇ 10000:1 fibroblasts and adipose-derived cells;
  • the method further comprises the steps of:
  • the seed cell-biomaterial complex is cultured in a bioreactor to obtain a tissue engineered tendon graft as described above.
  • the bioreactor is a pull-on tendon bioreactor disclosed in Application No. 200510110037.0.
  • the amount of seed cells in the graft is 1 ⁇ 10 5 cells/ml- 5 ⁇ 10 8 cells/ml.
  • the traction force on the seed cell-biomaterial composite in the bioreactor is 2-20N.
  • the biodegradable material is in the form of a cord.
  • a tissue engineered tendon graft as described above for use in the preparation of a graft for repairing a tendon defect. Accordingly, the present invention provides a tissue engineered human tendon constructed with new seed cells and constructed in vitro, and the tissue engineered tendon has good mechanical properties.
  • Figure 1 shows second generation human skin fibroblasts.
  • Figure 2 shows the second generation of human tendon cells.
  • Figure 3 shows primary adipose derived cells.
  • Figure 4 shows the results of identification of various phenotypes of adipose-derived cells;
  • A indicates the identification result of Viment in +
  • B indicates the identification result of CD 106 +
  • C indicates the identification result of CD34-
  • D indicates the identification result of CD29 + /CD49D +
  • E indicates the identification result of CD44 + /CD49D + .
  • Figure 5 shows the expression of type II collagen after adipose-derived cells are induced by cartilage.
  • Figure 6 shows the adipose-derived cells stained with red and stained with red to observe the formation of lipid droplets in the cytoplasm.
  • Figure 7 shows the formation of calcium nodules after osteogenic induction of adipose-derived cells.
  • Figure 8 shows the results of immunosuppressive regulatory function tests of adipose derived cells.
  • Figure 9 shows the pre-shaped bundle of PGA fiber scaffolds.
  • Figure 10 shows a PGA bracket with pre-formed bundle fibers attached to a "U" spring.
  • Figure 1 1 shows the appearance of the bioreactor.
  • Figure 12 shows the method of constructing tendon in a reactor with human dermal fibroblasts and/or adipose-derived stem cells.
  • Figure 13 shows the results of histological examination of tissue engineered tendons using human dermal fibroblasts as seed cells after static traction for 2 weeks + dynamic traction for 10 weeks;
  • A indicates skin fibroblasts + PGA; B indicates tendon cells + PGA
  • Figure 14 shows the results of biomechanical testing of tissue engineered tendons constructed in vitro under different tensions.
  • Figure 15 shows the results of histological examination of tissue engineered tendons using human adipose-derived stem cells as seed cells after static traction for 2 weeks + dynamic traction for 5 weeks.
  • tissue engineered tendon grafts adipose-derived cells or mixtures thereof can be used as seed cells for constructing tissue engineered tendon grafts, and a mixture of seed cells and biodegradable materials. Placed in a bioreactor for in vitro culture, tissue engineered human tendon with good physiological and mechanical properties can be obtained.
  • purified or isolated means that the purified or isolated material is substantially free of other cells, proteins or polypeptides.
  • xenograft refers to a method of removing a desired biological material, such as a tendon, from one species and reapplying to another species.
  • autologous transplantation refers to a method of removing a desired biological material, such as a tendon, from a patient and re-administering it to the same patient.
  • raft refers to a method of removing a desired biological material (such as tendon) from a body of the same species and applying it to another different patient.
  • biodegradable material As used herein, “pharmaceutically acceptable biodegradable material”, “medically acceptable biodegradable material”, “biodegradable material”, “biological material” and “material” are used in the same meaning.
  • the cell and growth factor are provided with a carrier, a material that has good biocompatibility with the cell or body tissue, and a rate of degradation that matches the growth of the tissue.
  • tissue engineered tendon graft As used herein, “tissue engineered tendon graft”, “tissue engineered tendon” and “tissue engineered tendon” are used interchangeably as long as they all represent a seed cell-biomaterial complex and can repair tendon defects in the body.
  • the transplant can be.
  • the seed cells which can be used in the tendon graft of the present invention are fibroblasts and/or adipose-derived cells, which may be derived from autologous or autologous, and fibroblasts are preferably derived from autologous. In the case of a mixture of the two, the ratio of the number of fibroblasts to adipose derived cells is 1: 10000-10000: 1, preferably 1: 5-100: 1, more preferably 1: 2-10: 1.
  • the source of the fibroblasts of the present invention is not particularly limited and may be fibroblasts of any origin.
  • the fibroblasts of the present invention are autologous (e.g., dermal, subcutaneous, and fibroblasts in other tissues), or the same Allogeneic fibroblasts (such as human fetal-derived fibroblasts).
  • the fibroblasts may also be fibroblasts derived from adipose stem cells, bone marrow stromal stem cells, or other stem cells.
  • the fibroblasts useful in the present invention are derived from the human body.
  • the seed cell is a human autologous dermal fibroblast.
  • Collagenase digestion and separation method Take the skin tissue under aseptic conditions, cut into 2 X 2 X 2 mm 3 size tissue blocks, rinse with phosphate buffer (PBS, containing 100 U/ml of cyanine and streptomycin) 2 Over the course, add two volumes of lmg/ml type II collagenase (Worthington, Freehold, NJ, USA), digest for 4 hours at 37 °C on a constant temperature shaker, then filter and centrifuge with 150 mesh nylon mesh, and precipitate the cells with PBS. The counts were counted by trypan blue staining to examine the viability of dermal fibroblasts, and the cells were cultured at a density of 1 x 107 disks (diameter 100 mm).
  • PBS phosphate buffer
  • Tissue block culture method Take the skin tissue under sterile anesthesia conditions, cut into 2 X 2 X 2 mm 3 size tissue blocks, rinse 2 times with phosphate buffer (PBS, containing 100 U/ml of cyan and streptomycin) . Place the tissue block evenly on the surface of the culture dish, place the culture dish in the incubator for 2-4 hours, gently add the culture solution to allow the liquid to slowly cover the small pieces of tissue, and wait for the cells to swim out.
  • PBS phosphate buffer
  • a preferred method is to culture dermal fibroblasts in a humidified, 5% CO 2 incubator. Suitable cultures include, but are not limited to: 1) DMEM medium ((Gibco) + 10% fetal bovine serum; 2) DMEM medium + 20% calf serum; 3) DMEM medium + 10-20 % autologous (allogeneic) human serum. Further, various growth factors (for example, cytokines for promoting fibroblast growth), various transgenic components, and various cellular components are added to the culture solution.
  • Dermal fibroblasts suitable for use in the present invention should be capable of proliferating in vivo or in vitro.
  • a preferred dermal fibroblast is a primary to fiftieth generation cell cultured in vitro, and immunohistochemical staining demonstrates expression of type I collagen, and in situ hybridization assay demonstrates expression of type I collagen mRNA.
  • ADCs adipose derivved cells obtained in adipose tissue also belong to a mesenchymal stem cell, which has multipotential differentiation potential with BMSC, and can be directed to bone under induction conditions. Cartilage, tendon and other directions. ADCs have other advantages over BMSCs. 1.
  • the source is abundant, easy to obtain, and the acquisition amount is much higher than that of BMSC, and the ADC cell proliferation ability is much larger than that of BMSC, which can provide sufficient cell volume for tissue engineering construction.
  • collagen secretion ability is strong, and a large amount of type I collagen in tendon tissue to maintain its good tensile strength, more suitable as tendon seed cells, more likely to form functional tendon tissue.
  • ADCs adipose-derived cells
  • tissue engineering tendons can not only be used as seed cells for tissue engineering tendons, but also have considerable practical value. Carrying out related research will likely form tissue engineered tendon products accepted by different individuals, breaking through tissue engineering tendon product individuals. The bottleneck of treatment.
  • a preferred method is as follows: The human adipose tissue discarded after liposuction is taken under aseptic conditions, transferred to a culture flask, and repeatedly washed with physiological saline, and an equal volume of 0.075% of type I collagenase (Worthington) is added.
  • adipose-derived cells were cultured in a saturated humidity, 5% CO 2 incubator. Suitable cultures include, but are not limited to: 1) DMEM medium ((Gibco) + 10% fetal bovine serum; 2) DMEM medium + 20% calf serum; 3) DMEM medium + 10-20 % autologous (allogeneic) human serum. Further, various kinds of growth factors (for example, cytokines for promoting growth of adipose-derived cells), various transgenic components, and various cellular components are added to the culture solution.
  • DMEM medium ((Gibco) + 10% fetal bovine serum
  • DMEM medium + 20% calf serum 3) DMEM medium + 10-20 % autologous (allogeneic) human serum.
  • growth factors for example, cytokines for promoting growth of adipose-derived cells
  • various transgenic components for promoting growth of adipose-derived cells
  • various cellular components are added to the culture solution.
  • Adipose-derived cells suitable for use in the present invention should be capable of proliferating in vivo or in vitro.
  • a preferred source of adipose cells are primary to thirtieth generation cells cultured in vitro. If necessary, some tendon cells and/or bone marrow stromal stem cells can be added to the seed cells. Methods of isolating and obtaining tendon cells are known in the art. L-5mg/ ⁇ (preferably about 1mg), a preferred method is to try the separation by trypsin, collagenase digestion, that is, the serum concentration of trypsin or collagenase is adjusted to 0. l-5mg / ml (preferably about 1mg) /ml), digestion at 37 °C ⁇ 2 °C in a constant temperature oscillator for about 4-20h.
  • a preferred method is to culture the tendon cells in a humidified, 5 % C 0 2 incubator. Suitable cultures include, but are not limited to: 1) DMEM medium ((Gibco) + 10% fetal bovine serum; 2) DMEM medium + 20% calf serum; 3) DMEM medium + 10-20 % autologous (allogeneic) human serum. Further, various growth factors (for example, cytokines for promoting tendon cell growth), various transgenic components, and various cellular components are added to the culture solution.
  • the source of bone marrow stromal stem cells (BMSCs) in the present invention is not particularly limited, and a preferred source is bone marrow derived from autologous.
  • a preferred source is bone marrow derived from autologous.
  • Methods for isolating bone marrow stromal stem cells are known in the art.
  • a preferred method is to extract autologous bone marrow from the bone marrow through anesthesia, and to separate the nucleated cells by density gradient centrifugation, and add a suitable culture solution (for example, containing 10% fetal bovine serum, L-glutamine 300 ug/ml).
  • Materials useful in the tissue engineered tendon of the present invention are pharmaceutically acceptable biodegradable materials including, but not limited to:
  • Degradable synthetic polymer materials such as poly- ⁇ -hydroxy acids (such as polylactic acid PLA, polyglycolic acid) PGA, polyhydroxybutyrate PHB, etc., polyanhydrides, polyphosphazenes, polyamino acids, pesudo-polyamino acids, polyorthoesters, Polyurethane (polyesterurethane), polycarbonate, polyethylene glycol, polyethylene oxide, polydioxanone, etc.;
  • Naturally degradable materials such as collagen, gelatin, glycosaminoglycan (GAGs), chitosan, chitin, alginate, calcium alginate Glue, etc.; various acellular matrix;
  • the bioreactor suitable for use in the present invention is not particularly limited, and various bioreactors conventionally used in the present invention can be used.
  • the preferred bioreactor has the following characteristics: 1.
  • the size and capacity are suitable, which facilitates the culture of cells or tissues and the discharge of metabolites, and is not easily contaminated. 2.
  • It can simulate the micro-environment of biomechanics such as tendon in the living body, and provide a growth environment suitable for tissue engineering tendons.
  • the parameters such as tension and frequency are controllable and easy to operate. Tissue engineering tendon
  • the present invention provides a novel tissue engineered tendon graft comprising: (a) a pharmaceutically acceptable biodegradable material; and (b) dermal fibroblasts and/or adipose derived cells, said Two seed cells are seeded separately or in combination with the biodegradable material.
  • the tissue engineered tendon graft provided by the invention has good mechanical properties and can be tested by methods conventional in the art, such as but not limited to the application of Intron biomechanical tester, B0SE biomechanical tester and the like to the tendon graft. The performance was measured.
  • the maximum tension of the tissue engineered tendon graft provided by the present invention at the time of testing is preferably from 10 to 80 N, more preferably from 40 to 80 N.
  • the tissue engineering tendon of the invention is relatively mature, and after a certain period of in vitro culture, most of the biomaterials are degraded, thereby avoiding tendon adhesion and breakage caused by a large amount of degradation products of biological materials when implanted early in the body. Cracks and other phenomena can repair tendon defects more effectively.
  • the tissue engineering tendon of the invention is relatively mature. After a certain period of in vitro culture, the seed cells secrete sufficient extracellular matrix such as collagen, so that the new tissue engineering tendon is implanted with certain biomechanical properties, and avoids early repair and repair. Force causes tendon rupture and the like.
  • the shape of the tissue engineered tendon graft of the present invention is not particularly limited, and may be arbitrarily shaped in accordance with the shape of the tissue defect, and the graft is usually elongated.
  • the seed cells expanded in vitro are inoculated into a biodegradable biomaterial which is biocompatible and can be absorbed by the body to form a seed cell-biomaterial composite, and the seed cell-biomaterial composite is placed in the bioreactor. After a certain period of cultivation, with the gradual degradation and absorption of biological materials, the seed cells continue to proliferate and secrete the matrix, replacing most of the biological materials to form relatively mature tissue engineering tendons, and when implanted into the defect site, the purpose of repairing the tendon defect can be achieved. .
  • Bioreactors commonly used in the art can be used as long as the necessary dynamic traction can be provided for the in vitro construction of the cell-material complex, preferably a pull-on tendon bioreactor.
  • the length and diameter of the tendon graft are determined according to the length and diameter of the tendon defect, and then cultured in vitro under stress conditions (2-20 Newtons) for one week, and once every two to two days to ensure cell nutrition, thereby forming a tendon for transplantation.
  • the seed cell concentration in the tissue engineered tendon of the present invention is usually about 1 X 10 5 /ml to 5 X 10 8 /ml or higher, preferably IX 10 6 /ml to IX 10 8 /ml, more preferably 5 X 10 6 /ml to 5 X 10 7 /ml.
  • the seed cell concentration is usually adjusted with the culture medium and then mixed with the degradable material.
  • the ratio of the culture solution to the degradable material during mixing is not particularly limited, but the maximum amount of the culture solution which can be adsorbed by the material is suitable.
  • tissue engineering tendon graft of the present invention various other cells, growth factors, and various transgenic components may be added or compounded to maintain the tendon cell phenotype, promote tendon cell growth, and promote tissue engineering tendon in vivo.
  • the tissue engineered tendon graft or tendon formed by the method of the present invention can be directly implanted into the tendon defect in the body and repair the tendon defect.
  • the dermal fibroblasts and/or adipose-derived cells are widely distributed and easy to obtain, which solves the problem of insufficient source of seed cells for tissue engineered tendon construction.
  • Adipose-derived cells have low immunogenicity and immunoregulatory functions, and are expected to solve the problem of immune rejection in allograft repair.
  • a tissue which can be arbitrarily shaped in accordance with the shape of a tissue defect can survive for a lifetime in a living body.
  • the foreskin tissue discarded during the operation was cut into 2 X 2 X 2 mm 3 tissue blocks, and the phosphate buffer solution (PBS, containing 100 U/ml of cyanine and streptomycin) was washed twice, and the volume was added twice.
  • PBS phosphate buffer solution
  • the lmg/ml type II collagenase (purchased from Worthington, Freeho ld, NJ, USA) was placed in a 37 ° C shaker for 4 h and then centrifuged through a 150 mesh nylon mesh sieve. The precipitated cells were washed twice with PBS.
  • the trypan blue staining was used to examine the fibroblast viability, and the cells were cultured at a density of IX 107 (the diameter of the culture dish was 100 mm).
  • the culture medium was DMEM (purchased from Gibco, Gland, Island, NY, USA) contains 10% fetal bovine serum, L-glutamine 300ug/ml, vitamin C50ug/ml, cyan and streptomycin 100U/ml each, fibroblasts passed to the second generation, via 0.25 % trypsin digestion was used to collect cells for experiments ( Figure 1).
  • DMEM purchased from Gibco, Gland, Island, NY, USA
  • the viability of the tendon cells was examined by trypan blue staining, and the cells were cultured at a density of IX 107 (100 mm diameter of the culture dish).
  • the culture medium was DMEM (purchased from Gibco, Gland, Island, NY). , USA) containing 10% fetal bovine serum, L-glutamine 300ug/ml, vitamin C50ug/ml, cyan and streptomycin 100U/ml each, tendon cells passed to the second generation, collected by 0.25% trypsin Used for experiments ( Figure 2).
  • DMEM purchased from Gibco, Gland, Island, NY. , USA
  • the human tendon tissue discarded after liposuction was taken under aseptic conditions, transferred to a culture flask, washed repeatedly with physiological saline, and an equal volume of 0.075% type I collagenase (purchased from Worthington, Freehold, NJ, USA) was added. Digested at 37 ° C with a constant temperature shaker, and centrifuged at 300 g for 10 minutes, respectively, to obtain a high-density cell pellet, discard the supernatant and floating adipose tissue, and shake the cells. After counting, the plate was densified at 1 ⁇ 107.
  • the cells were cultured in a DMEM (purchased from Gibco, Gland, Island, NY, USA) containing 10% fetal calf serum, and cultured at 37 ° C, 5% CO 2 , 100% saturated humidity. After 24 hours, the medium was changed for the first time, and the floating cells were repeatedly washed with PBS, and the culture solution was added, and passage was carried out while the cells were substantially overgrown at the bottom of the culture dish (Fig. 3). The cells were passed to the desired passage, and the cells were collected by 0.25% trypsinization for experiments.
  • DMEM purchased from Gibco, Gland, Island, NY, USA
  • adipose-derived cells were cultured on cell slides, and fixed to 4% paraformaldehyde for 15 minutes at 70% confluence. Add various antibodies to the surface antigen to be tested, incubate at 37 °C for 1 hour, rinse with PBS, and add FITC. The secondary antibody (DAK0, Carpinteria, US A) was incubated at 37 °C for 30 minutes, rinsed again with PBS, and cored with Heochest 33258 (purchased from Sigma (USA) or Propidium Iodide (purchased from Sigma (USA)) The expression of cell surface antigen was observed under a laser confocal microscope. Multi-directional differentiation potential of adipose-derived cells
  • the third generation cells were induced by cartilage in a micro-branche culture method.
  • the final concentration of the inducing factor was TGF- ⁇ ⁇ (purchased from R&D Company (USA)) 10 ng/mL, IGF (purchased from R&D Company (USA)) 100 ng/mL.
  • Dex purchased from Sigma (USA)) 0.1 ym O l/L, transferrin (purchased from Sigma (USA)) 6.25 ⁇ g/mL.
  • the third generation of cells was cultured for 7 days for fat induction, and the inducing solution was base medium and 0.5 mmol/L IBMX (purchased from Sigma (USA)), 1 ⁇ mol/L Dex, 10 ⁇ mol/L insulin (purchased from Sigma (USA), 200 ⁇ mol/L indomethacin (purchased from Sigma (USA)), observed changes in cell morphology under an inverted phase contrast microscope, and induced for 14 days.
  • IBMX purchased from Sigma (USA)
  • 1 ⁇ mol/L Dex 1 ⁇ mol/L Dex
  • 10 ⁇ mol/L insulin purchasedd from Sigma (USA)
  • 200 ⁇ mol/L indomethacin purchasedd from Sigma (USA)
  • the third-generation cell culture was subjected to osteogenic induction at 7 days, and the inducing liquid component was added to the basal medium to add 2.16 g/L ⁇ -glycerol phosphate, lOnmol/L vitamin D3. For 20 days of induction, specific mineralization was performed on the mineralized nodules.
  • the material fibers are pre-formed into a bundle (Fig. 9) and fixed on a "U” spring to give the stent a constant static tension (Fig. 10), and then immersed in 75% ethanol for 1 hour and then rinsed with PBS. After all, the UV lamp is disinfected and air-dried for use.
  • Example 5
  • Skin fibroblasts and/or adipose-derived cells were passed to passages 2 to 4, and cells were collected by 0.25% trypsinization to prepare a 2.0 ⁇ 107 ml cell suspension. Inoculate the cell suspension onto the pre-made PGA fiber scaffold, first place the cell-material complex in a carbon dioxide incubator for 4 hours, then add about 30 ml of DMEM medium containing 10% fetal bovine serum, and continue to place in the incubator. Internal culture. The culture medium is changed once every two days to ensure cell nutrition. After about 2 weeks, the cells secrete enough extracellular matrix and transfer to the cell culture chamber of the pull-type tendon bioreactor for dynamic stretch culture. After a certain period of time (preferably 3 to 10 weeks), the cells can be used.
  • Tissue engineered tendon graft for tendon defect repair Bioreactor loaded in species
  • the traction force on the daughter cell-biomaterial composite is 2-20N
  • the pulling frequency is 1 ⁇ 12 hours per day
  • the stretching is performed for 2 ⁇ 30 seconds each time
  • the interval is 5 ⁇ 60 seconds
  • the displacement is the length of the graft. 5 to 30%.

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Description

组织工程肌腱及其体外构建方法 技术领域
本发明涉及医学和生物医学工程领域， 更具体地涉及应用真皮成纤维细胞和 (或） 脂肪来源细胞在体外构建组织工程肌腱的制备方法和用途。 背景技术
肌腱是连接骨骼肌肌腹与骨之间的致密胶原纤维结缔组织束，是骨骼肌的组成 部分。 肌腱的结构可以看成是肌肉的延续、 退化部分。 新鲜标本的肌腱呈银白色、 索带状， 质地坚韧。 在松弛状态下表面有波纹， 如果牵拉超过松弛状态的 4%时， 这波纹便消失， 张力取消后波纹再现。 在肌 -腱联接处， 胶原纤维进入肌内膜， 附 着在肌纤维的肌膜上在腱 -骨连接处， 腱束直接连续到骨和骨膜上， 其中绝大部分 胶原纤维进入骨，形成 Sharpey穿纤维。肌腱传导肌腹收缩所产生的力，牵引骨骼， 使之产生运动。 肌腱本身不具有收缩能力， 但能抵抗很大的张力。
肌腱缺损是目前临床工作中经常遇到的问题， 对于肌腱缺损的治疗方法主要 是： 1、 自体肌腱移植。 2、 同种异体肌腱移植。 3、 肌腱移植替代物。 但以上方法 均存在着某些弊端， 自体肌腱移植存在肌腱供区缺乏的问题。新鲜的异体肌腱移植 会引起严重的免疫排斥反应，经过冻干处理后的同种异体肌腱保存的仅仅是胶原纤 维， 将来仍需要自体肌腱细胞进行替代。人工肌腱替代物虽然近期生物力学强度较 好， 但远期终会发生降解， 而且还会引起炎症、 纤维化和肌腱粘连等反应甚至替代 物排出体外。
上世纪 80年代末期， 组织工程学的兴起和蓬勃发展为解决这一难题提供了可 能。 曹谊林等于 1994年首次报道了用聚羟基乙酸（PGA )短纤维在裸鼠皮下构建出 了组织工程肌腱样组织。刘永涛等则在有完善免疫功能的鸡趾指屈肌腱原位， 应用 自体肌腱细胞构建组织工程肌腱成功， 但是， 应用自体肌腱细胞作为组织工程肌腱 的种子细胞仍需切取较大的肌腱组织， 造成新的损伤。 因此， 寻找新的种子细胞来 源成为组织工程肌腱发展的首要问题。
此外， 目前国内外组织工程肌腱的研究主要集中在动物体内的修复实验， 即在 体外扩增出足够数量的种子细胞，与生物材料复合后立即或仅在体外培养一至两周 即植入体内缺损部位， 尽管获得了一些初步的成功， 但这种将接种细胞后的支架材 料直接回植入体内形成肌腱的方式仍然存在一些重大缺陷： 包括： （1 ) 植入的是 细胞材料复合物而非肌腱移植物， 导致种子细胞存活不佳， 从而引起移植失败， 无 法获取恒定的高成功率。 （2 ) 植入的支架为尚未降解的材料， 其分解产物多为酸 性， 可以直接导致炎性反应， 瘢痕形成和肌腱粘连， 从而直接影响了肌腱的修复效 果。 （3 ) 由于植入的是非肌腱移植物， 随着材料的降解， 细胞 -材料复合物的力学 性能大大降低， 无法修复高张力部位的肌腱缺损。 因此， 虽然有较多的肌腱构建和 修复的动物体内实验， 但由于上述问题的存在， 离临床应用仍有相当的距离。
由此可见， 采用组织工程技术构建肌腱组织、 修复肌腱组织缺损， 并达到产业 化生产的目的， 关键之一是获取大量有正常功能的肌腱细胞。但是肌腱细胞经多次 传代后丧失分泌基质的能力，难以获得大量有功能活性的肌腱细胞来修复大块缺损 组织。关键之二是要构建出具有一定力学性能、大部分材料已发生降解的相对成熟 的肌腱组织。 因此， 探索更广泛的种子细胞来源、 更优化的体外构建技术已成为组 织工程肌腱研究的焦点。
针对关键问题一， 许多学者作了大量研究工作， 试图拓展肌腱组织工程种子细 胞来源， 并促进其增殖。
第一， 寻找可替代细胞例如骨髓基质干细胞（Mesenchymal stem ce l l MSC)、 真皮成纤维细胞 （Dermal Fibrob last DF ) 。 MSC是一群具有多相分化潜能的细胞， 在特定的诱导条件下可分化为多种间充质组织细胞， Awad等分离、 培养 MSC后与 胶原复合形成细胞胶原复合物植入人工缺损髌韧带处， 与对照组（单纯应用胶原） 进行比较， 发现实验组组织的弹性模量、 最大张力、 硬度等较对照组大， 而在组织 学和形态学方面两者并没有很大差别。 然而， 骨髓基质干细胞的来源有限， 分离较 为复杂。陈兵等人用猪的自体成纤维细胞成功构建组织工程肌腱并修复趾浅屈肌腱 缺损， 证明了真皮成纤维细胞作为组织工程肌腱或韧带构建的种子细胞的可行性。
第二， 促进种子细胞增殖与基质合成。生长因子是通过细胞间信号传递影响细 胞活动的一类多肽因子， 在体内或体外可促进或抑制细胞增殖。许多学者直接使用 生长因子研究并证实其在体内体外对肌腱损伤修复的促进作用。此外， 还有人利用 转基因技术， 将基因信息传递给细胞以此来改变细胞功能。利用此种技术使靶细胞 增加或抑制合成和分泌蛋白质（如生长因子），调控细胞的生长，参与组织修复过程。 成功应用这一方法可使细胞、组织按要求合成分泌细胞因子以调控其生长。 已有学 者成功地用不同方法将基因转入肌腱中， LacZ 标记基因被应用于髌韧带后持续表 达了 6周， 在鸡趾屈肌腱的实验中， Lou等运用转基因技术将 LacZ标记基因转入 肌腱中， 75 天后还可以检测到该基因， 并提出了减少和阻止肌腱损伤后的粘连形 成的一些方法。
虽然经过上述各种研究， 仍没有彻底解决肌腱种子细胞来源缺乏的难题。 针对关键问题二， 有学者尝试在体外应用多种生物材料进行肌腱组织的构建。 曹德君等人利用可吸收生物材料聚羟基乙酸（po lyglycol ic acids , PGA) 和肌腱细 胞在体外构建组织工程肌腱， 用 U形弹簧给予细胞 -PGA复合物持续张力后体外培 养， 6周后形成与正常肌腱结构相似、 具有一定力学性能的肌腱样组织， 但此种方 法无法精确计量所施加的张力大小及频率等参数，所构建的组织的力学性能极为薄 弱。 体外模拟体内微环境仍需要进一步的改进和优化。
因此， 本领域迫切需要一种用新的种子细胞并在体外构建的组织工程化人肌 腱， 而且所述的组织工程化肌腱具有良好的力学性能。 发明内容
本发明旨在提供一种组织工程化人肌腱移植物。
本发明的另一个目的是提供所述的组织工程化肌腱移植物的体外制备方法。 本发明的再一个目的是提供上述组织工程化肌腱移植物的用途。 在本发明的第一方面， 提供了一种组织工程化肌腱移植物， 它包括：
(a)药学上可接受的生物可降解材料； 和
(b)种子细胞， 所述的种子细胞接种于所述的生物可降解材料， 且种子细胞 选自下组： （i )成纤维细胞， （i i )脂肪来源细胞， 或（i i i ) 1 : 10000— 10000 : 1 的成纤维细胞和脂肪来源细胞的混合物。
在另一优选例中， 所述组织工程化肌腱移植物的最大张力为 10— 80N。
在另一优选例中， 所述的种子细胞是成纤维细胞或成纤维细胞和脂肪来源细 胞的混合物。
在另一优选例中， 所述的种子细胞的含量为 1 X 105个细胞 /ml-5 X 108个细胞
/ml。
在另一优选例中， 所述的生物可降解材料为条索状。
在另一优选例中， 所述的成纤维细胞和脂肪来源细胞来源于自体或同种异 体。
在另一优选例中，所述的药学上可接受的生物可降解材料选自下组：聚乳酸、 聚羟基乙酸、 聚羟基丁酸、 聚酸酐、 聚偶磷氮、 聚氨基酸、 假聚氨基酸、 聚原酸 酯、 聚酯尿烷、 聚碳酸酯、 聚乙二醇、 聚环氧乙烷、 聚对二氧六环酮、 胶原、 明 胶、 糖氨聚糖、 壳聚糖、 甲壳素、 海藻酸盐、 藻酸钙凝胶、 脱细胞基质， 及其各 种类型和比列的混合物。 在本发明的第二方面， 提供了一种制备上述的组织工程化肌腱移植物的方 法， 包括步骤：
将种子细胞与药学上可接受的生物可降解材料混合， 得到种子细胞一生物材 料复合物， 其中所述的种子细胞接种于所述的生物可降解材料， 且种子细胞选自 下组： （i) 成纤维细胞， （ii) 脂肪来源细胞， 或 （iii) 1:10000〜10000:1的 成纤维细胞和脂肪来源细胞的混合物；
在另一优选例中， 它还包括步骤：
将种子细胞一生物材料复合物在生物反应器中培养得到如上所述的组织工 程化肌腱移植物。
在另一优选例中， 所述的生物反应器是申请号为 200510110037.0 中所公开 的一种牵拉式肌腱生物反应器。
在另一优选例中， 移植物中种子细胞的含量为 1X105个细胞 /ml-5X108个细 胞 /ml。
在另一优选例中， 生物反应器中加载在种子细胞一生物材料复合物上的牵引 力为 2— 20N。
在另一优选例中， 所述的生物可降解材料为条索状。 在本发明的第三方面， 提供了一种如上所述的组织工程化肌腱移植物的用 途， 它可用于制备修复肌腱缺损的移植物。 据此， 本发明提供了一种用新的种子细胞并在体外构建的组织工程化人肌腱， 而且所述的组织工程化肌腱具有良好的力学性能。 附图说明
图 1显示了第二代人皮肤成纤维细胞。
图 2显示了第二代人肌腱细胞。
图 3显示了原代脂肪来源细胞。 图 4显示了对脂肪来源细胞各种表型的鉴定结果； 其中
A表示 Viment in+的鉴定结果， B表示 CD 106+的鉴定结果， C表示 CD34—的鉴定 结果， D表示 CD29+/CD49D+的鉴定结果， E表示 CD44+/CD49D+的鉴定结果。
图 5显示了脂肪来源细胞成软骨诱导后， II型胶原的表达情况。
图 6 显示了脂肪来源细胞成脂肪诱导后， 由红染色， 观察细胞浆内脂滴形成 的情况。
图 7显示了脂肪来源细胞成骨诱导后， 钙结节形成的情况。
图 8显示了脂肪来源细胞的免疫抑制调控功能检测结果。
图 9显示了预塑形后的束状 PGA纤维支架。
图 10显示了预塑形的束状纤维固定在 " U " 形弹簧上的 PGA支架。
图 1 1显示了生物反应器外观。
图 12 显示了人真皮成纤维细胞和 /或脂肪来源干细胞在反应器内构建肌腱方 法。
图 13显示了应用人真皮成纤维细胞为种子细胞，静态牵拉 2周 +动态牵拉 10 周后的组织工程化肌腱组织学检测结果； 其中
A表示皮肤成纤维细胞 +PGA ; B表示肌腱细胞 +PGA
图 14显示了不同张力下体外构建组织工程肌腱的生物力学检测结果。
图 15显示了应用人脂肪来源干细胞为种子细胞， 静态牵拉 2周 +动态牵拉 5 周后的组织工程化肌腱组织学检测结果。 具体实施方式
发明人经过广泛而深入的研究， 意外地发现可以将人的真皮成纤维细胞、 脂肪来 源细胞或其混合物作为构建组织工程化肌腱移植物的种子细胞， 并将种子细胞和生物 可降解材料的混合物放置在生物反应器中进行体外培养， 能获得具有良好生理力学性 能的组织工程化人肌腱。 术语
术语 "纯化的或分离的"指纯化的或分离的物质基本上不含有其他细胞、 蛋 白质或多肽。
术语 "异种移植"指将所需生物材料（如肌腱）从某一物种中取出并再施用于 另一物种对象的方法。 术语 "自体移植"指将所需生物材料（如肌腱）从某病人中取出并再施用于同 一病人的方法。
术语 "异体移植" 指将所需生物材料 (如肌腱)从同一物种的某个体中取出并 再施用于另一不同病人的方法。
如本文所用 "药学上可接受的生物可降解材料" 、 "医学上可接受的生物可 降解材料" 、 "生物可降解材料" 、 "生物材料" 和 "材料" 是同样的含义， 都 表示可给细胞及生长因子提供一个载体， 与细胞或机体组织具有良好的生物相容 性， 而且具有与组织生长相匹配的降解速率的材料。
如本文所用， "组织工程化肌腱移植物" 、 "组织工程化肌腱" 和 "组织工 程肌腱" 可互换使用， 只要它们都表示一种含有种子细胞一生物材料复合物并能 修补体内肌腱缺损的移植物即可。 种子细胞
可用于本发明肌腱移植物的种子细胞是成纤维细胞和 （或） 脂肪来源细胞， 这两种细胞可以来自自体， 也可以来自异体， 成纤维细胞取自自体更佳。 若为两者 的混合物， 则成纤维细胞和脂肪来源细胞的数量之比为 1 : 10000-10000 : 1， 较佳地 为 1 : 5-100 : 1， 更佳地为 1 : 2-10 : 1。
本发明的成纤维细胞的来源没有特别限制， 可以是任何来源的成纤维细胞， 通常， 本发明的成纤维细胞是自体的（如真皮、 皮下组织以及其他组织中的成纤 维细胞）、 或同种异体的成纤维细胞（如人胎儿来源的成纤维细胞）。 成纤维细胞 还可以是衍生自脂肪干细胞、 骨髓基质干细胞、 或其他干细胞的成纤维细胞。
可用于本发明的成纤维细胞来自人体。
尽管自体的成纤维细胞是优选的， 但异体的成纤维细胞的来源也可使用。 在本发明的一个优选例中， 所述的种子细胞是人自体真皮成纤维细胞。
分离和获得真皮成纤维细胞的方法是本领域中熟知的。 常用的分离方案包括：
(a)胶原酶消化、 分离法： 在无菌条件下取皮肤组织， 切成 2 X 2 X 2mm3大小组 织块，磷酸缓冲液（PBS，含青、链霉素各 100U/ml)冲洗 2遍，加入二倍体积的 lmg/ml I I型胶原酶（Worthington, Freehold, NJ， USA)，置于 37 °C恒温摇床消化 4h后用 150 目尼龙网筛过滤离心， 沉淀细胞用 PBS洗 2次计数， 台盼蓝染色检查真皮成纤维细 胞活力， 以 1 X 107盘密度（培养皿直径 100mm)培养细胞。 (b)组织块培养法： 在麻醉无菌条件下取皮肤组织， 切成 2 X 2 X 2mm3大小组织 块， 磷酸缓冲液（PBS， 含青、 链霉素各 100U/ml)冲洗 2遍。 将组织块在培养皿表 面均匀摆置， 将培养皿放置于培养箱中放置 2-4小时， 轻轻加入培养液让液体缓慢 覆盖组织小块， 等待细胞游出。
真皮成纤维细胞的培养方法和培养液也是本领域中熟知的。 一种优选的方法 是将真皮成纤维细胞在饱和湿度、 5%C02培养箱内培养。合适的培养液包括（但并不 限于）： 1) DMEM培养基（（Gibco公司） +10%胎牛血清； 2) DMEM培养基 +20%小牛血清； 3) DMEM培养基 + 10-20%自体（异体）人血清。 此外， 上述培养液中添加各种生长因子 (例如促进成纤维细胞生长的细胞因子等）、 各种转基因成分、 各种细胞成分。
适用于本发明的真皮成纤维细胞应能在体内或体外增殖。 一种优选的真皮成 纤维细胞是体外培养的原代至第五十代细胞，且免疫组织化学染色证明 I型胶原表 达， 原位杂交检测证明 I型胶原 mRNA表达。 本发明人研究发现， 脂肪组织中获得的脂肪来源细胞 （adipose deri ved ce l l s, ADCs ) 也属于一种间充质干细胞， 与 BMSC—样具有多向分化潜能， 能够在 诱导条件下向骨， 软骨， 肌腱等方向分化。 与 BMSC相比 ADC还具有其它优点。 1、 来源丰富， 易于获取， 且获取量要远高于 BMSC, 而且 ADC细胞增殖能力也远大于 BMSC , 能够为组织工程化构建提供充足的细胞量。 2、 胶原分泌能力较强， 而肌腱 组织中需要大量的 I型胶原维持其良好的抗拉强度， 更适合作为肌腱种子细胞， 更 容易形成功能性肌腱组织。 3、 具有低免疫原性和免疫调节功能， 更适合应用于同 种异体间的移植， 具有更广泛的临床应用前景。
因此， 脂肪来源细胞 （ADC ) 不仅能够作为组织工程肌腱的种子细胞， 而且具 有相当大的实用价值，开展相关的研究将有可能形成被不同个体接受的组织工程肌 腱产品， 突破组织工程肌腱产品个体化治疗的瓶颈问题。
分离和获得脂肪来源细胞的方法是本领域中已知的。 一种优选的方法是： 在无菌条件下取吸脂术后废弃的人脂肪组织， 转移到培养瓶中， 用生理盐水 反复冲洗，加入等体积的 0. 075%的 I型胶原酶（Worthington, Freeho l d, NJ， USA)， 置于 37 °C恒温摇床消化， 分别于 1小时后， 300g离心 10分钟， 获得高密度的细胞 沉淀物， 弃上清和漂浮的脂肪组织， 细胞振匀， 计数后以 1 X 107盘密度（培养皿直 径 100mm)培养细胞。
脂肪来源细胞的培养方法和培养液也是本领域中熟知的。 一种优选的方法是 将脂肪来源细胞在饱和湿度、 5%C02培养箱内培养。 合适的培养液包括（但并不限 于）： 1) DMEM培养基（（Gibco 公司） +10%胎牛血清； 2) DMEM培养基 +20%小牛血清； 3) DMEM培养基 + 10-20%自体（异体）人血清。 此外， 上述培养液中添加各种生长因子 (例如促进脂肪来源细胞生长的细胞因子等）、 各种转基因成分、 各种细胞成分。
适用于本发明的脂肪来源细胞应能在体内或体外增殖。 一种优选的脂肪来源 细胞是体外培养的原代至第三十代细胞。 如果必要， 可以在种子细胞中加入一些肌腱细胞和 /或骨髓基质干细胞。 分 离和获得肌腱细胞的方法是本领域中已知的。 一种优选的方法是通过胰酶、 胶原 酶消化进行分离， 即用无血清 DMEM培养基（GIBC0公司）将胰酶或胶原酶浓度调整 至 0. l-5mg/ml (较佳地约为 lmg/ml)， 37 °C ± 2 °C恒温振荡器内消化约 4_20h。
肌腱细胞的培养方法和培养液也是本领域中熟知的。 一种优选的方法是将肌 腱细胞在饱和湿度、 5 % C 02培养箱内培养。合适的培养液包括（但并不限于）： 1 ) D M E M 培养基（（Gibco公司） +10%胎牛血清； 2) DMEM培养基 +20%小牛血清； 3) DMEM培养 基 + 10-20%自体（异体）人血清。 此外， 上述培养液中添加各种生长因子（例如促进 肌腱细胞生长的细胞因子等）、 各种转基因成分、 各种细胞成分。
本发明中骨髓基质干细胞（BMSCs)的来源没有特别限制， 一种优选的来源是 来自自体的骨髓。 分离获得骨髓基质干细胞的方法是本领域中已知的。 一种优选 的方法是麻醉下骨髓穿剌抽取自体骨髓， 以密度梯度离心法分离出其中的有核细 胞， 加入适合的培养液（如含有 10%胎牛血清， L-谷氨酰胺 300ug/ml， 维生素 C50ug/ml , 青、 链霉素各 100U/ml， 地塞米松 5nM (Sigma)的 DMEM (Gibco， Gland I sland, NY, USA)条件培养液）， 制成细胞悬液， 以约 1 X 105/cm2的密度接种于培 养皿， 于 37 °C、 5%C02、 100%饱和湿度的条件下培养。 48小时后首次换液， 加入条 件培养液继续培养隔日换液。 待细胞生长近汇合后， 以 0. 25%胰蛋白酶 +0. 02%EDTA 消化， 以 l X 104/cm2的密度接种进行细胞传代。 生物可降解材料
可用于本发明的组织工程化肌腱的材料是药学上可接受的生物可降解材料， 包括（但并不限于）：
(a)可降解性合成高分子材料， 例如聚 α -羟基酸（如聚乳酸 PLA、聚羟基乙酸 PGA 、 聚 羟 基丁 酸 PHB 等 ） 、 聚 酸 酐 （polyanhydrides) 、 聚 偶 磷氮 (polyphosphazenes) 、 聚 氨 基 酸 (polyamino acid) 、 假 聚 氨 基 酸 (pesudo-polyamino acid) 、 聚 原 酸 酉旨 (polyorthoesters) 、 聚 酉旨 尿 焼 (polyesterurethane) 聚碳酸酉旨（polycarbonate)、 聚乙二醇、 聚环氧乙烷、 聚 对二氧六环酮（polydioxanone)等；
(b)天然可降解材料， 例如胶原（collagen)、 明胶（gelatin)、 糖氨聚糖 (glycosaminoglycan, GAGs)、 壳聚糖（chitosan)、 甲壳素（chitin)、 海藻酸盐、 藻酸钙凝胶等； 各种脱细胞基质；
(c)上述材料的混合物或复合材料， 尤其是高分子材料与天然材料的复合材 料。
其中优选聚羟基乙酸 PGA、 聚乳酸 PLA， 或两者共纺物 PGLA等。 生物反应器
适用于本发明的生物反应器没有特别限制， 可以使用本发明常用的各种生物 反应器。 优选的生物反应器具有以下特征： 1、 大小、 容量合适， 利于细胞或组织 的培养和代谢产物的排出， 且不易污染。 2、 能够模拟生物体内肌腱的生物力学等 微环境， 提供适于组织工程肌腱的生长环境。 3、 拉力、 频率等参数可控， 且便于 操作。 组织工程肌腱
本发明提供了一种新的组织工程化肌腱移植物， 它包括： （a)药学上可接受的 生物可降解材料； 和（b)真皮成纤维细胞和 （或） 脂肪来源细胞， 所述的两种种子 细胞分别或混合接种于所述的生物可降解材料。本发明提供的组织工程化肌腱移植 物具有良好的力学性能， 可以使用本领域常规的方法进行测试， 例如但不限于应用 Intron生物力学测定仪， B0SE生物力学测试仪等对肌腱移植物的抗拉性能进行测 定。
测试时本发明提供的组织工程化肌腱移植物的最大张力较佳的为 10— 80N，更 佳的为 40— 80N。
本发明的组织工程肌腱相对成熟， 在体外培养一定时期后， 生物材料大部分 降解， 避免了早期回植入体内时， 因生物材料的大量降解产物引发的肌腱粘连、 断 裂等现象， 能够更有效的修复肌腱缺损。
本发明的组织工程肌腱相对成熟， 在体外培养一定时期后， 种子细胞分泌了 充分的胶原等细胞外基质，使新生组织工程肌腱植入时已经具备一定的生物力学性 能， 避免了修复早期因受力导致肌腱断裂等。
本发明的组织工程肌腱移植物的形状没有特别限制， 可以按照组织缺损的形 状任意塑形， 通常移植物为长条形。 体外培养扩增的种子细胞接种到生物相容性良好并可被机体吸收的可降解生 物材料上形成种子细胞一生物材料复合物，将这一种子细胞一生物材料复合物置于 生物反应器中， 培养一定时期后， 随着生物材料的逐渐降解吸收， 种子细胞继续增 殖并分泌基质， 替代大部分生物材料而形成相对成熟的组织工程肌腱， 植入到缺损 部位时， 可达到修复肌腱缺损的目的。
可以使用本领域常用的生物反应器， 只要可以为细胞一材料复合物的体外构 建提供必要的动态牵引力， 优选牵拉式肌腱生物反应器。 将肌腱移植物的长度、 直径依照肌腱缺损长度与直径确定， 然后在应力条件（2-20 牛顿）下体外培养一 周， 每一至二天换液一次， 以保证细胞营养， 从而形成供移植的肌腱。
本发明组织工程肌腱中的种子细胞浓度通常约为 l X 105/ml至 5 X 108/ml或更 高， 较佳地为 I X 106/ml至 I X 108/ml， 更佳地为 5 X 106/ml至 5 X 107/ml。 通常以 培养液调整种子细胞浓度， 然后与可降解材料混合。混合时培养液与可降解材料的 比例没有特别限制， 但是以该材料能够吸附的培养液最大量为宜。
此外， 在本发明的组织工程肌腱移植物中， 还可添加或复合其他各种细胞、 生长因子、 各种转基因成分， 从而保持肌腱细胞表型、 促进肌腱细胞生长， 以及促 进组织工程肌腱在体内的血管神经化。 用本发明方法形成的组织工程肌腱移植物或肌腱， 可直接植入体内肌腱缺损 处并修复肌腱缺损。 本发明提到的上述特征， 或实施例提到的特征可以任意组合。 本案说明书所揭示 的所有特征可与任何组合物形式并用， 说明书中所揭示的各个特征， 可以任何可提供 相同、 均等或相似目的的替代性特征取代。 因此除有特别说明， 所揭示的特征仅为均 等或相似特征的一般性例子。 本发明的主要优点在于：
( 1)真皮成纤维细胞和 （或） 脂肪来源细胞分布广泛， 取材容易， 解决了组织 工程化肌腱构建种子细胞来源不足的问题。
(2)脂肪来源细胞具有低免疫原性和免疫调节功能， 有望解决同种异体移植修 复的免疫排斥问题。
(3)可以按照组织缺损的形状任意塑形形成的组织可以在生物体内终生存活。
(4)利用新型的生物反应器提供精确可调控的单向牵引力， 进行肌腱构建， 并 利用 exc luded vo lume effect (EVE)效应来加速肌腱的成熟。
(5)体外形成肌腱后， 细胞处于自然细胞外基质的包绕中， 再回植体内可以避 免细胞死亡， 从而获得相对恒定的高成功移植率。
(6)在体外构建的肌腱再植入体内时， 由于支架材料已经大部降解， 避免了酸 性降解产物的产生， 从而可以防止肌腱纤维化和粘连。
(7)在体外构建出具有一定生物力学性能的组织工程肌腱， 回植后可以像正常 肌腱移植一样开展早期的功能锻炼， 有助于防止肌腱粘连和促进功能康复。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法， 通 常按照常规条件或按照制造厂商所建议的条件。除非另外说明， 否则所有的百分比 和份数按重量计。
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉 的意义相同。此外， 任何与所记载内容相似或均等的方法及材料皆可应用于本发明 方法中。 文中所述的较佳实施方法与材料仅作示范之用。 实施例 1
皮肤成纤维细胞的分离与培养
在无菌条件下取术中废弃的包皮组织， 切成 2 X 2 X 2mm3大小组织块， 磷酸缓 冲液（PBS， 含青、 链霉素各 100U/ml )冲洗 2遍， 加入二倍体积的 lmg/ml 的 I I型 胶原酶（购自 Worthington, Freeho l d, NJ， USA)，置于 37 °C恒温摇床消化 4h后用 150 目尼龙网筛过滤离心， 沉淀细胞用 PBS洗 2次计数， 台盼蓝染色检查成纤维细胞活 力， 以 I X 107盘密度（培养皿直径 100mm)培养细胞， 培养液为 DMEM (购自 Gibco, Gland, Island, NY, USA)含 10%胎牛血清， L -谷氨酰胺 300ug/ml,维生素 C50ug/ml, 青、 链霉素各 100U/ml， 成纤维细胞传至第二代， 经 0.25%胰酶消化收集细胞用于 实验（图 1)。 实施例 2
肌腱细胞的分离与培养
在无菌条件下取新鲜截肢术后废弃的人肌腱组织， 切成 2X2X2mm3大小组织 块，磷酸缓冲液（PB S，含青、链霉素各 10 OU/m 1 )冲洗 2遍，加入二倍体积的 0.25mg/m 1 的 II型胶原酶（购自 Worthington, Freehold, NJ, USA) , 置于 37°C恒温摇床消化， 分别于 4、 5、 7小时后， 用 150 目尼龙网筛过滤离心， 沉淀细胞用 PBS洗 2次计数， 台盼蓝染色检查肌腱细胞活力， 以 IX 107盘密度（培养皿直径 100mm)培养细胞， 培养液为 DMEM (购自 Gibco, Gland, Island, NY, USA)含 10%胎牛血清， L-谷氨酰 胺 300ug/ml， 维生素 C50ug/ml， 青、 链霉素各 100U/ml， 肌腱细胞传至第二代， 经 0.25%胰酶消化收集细胞用于实验（图 2)。 实施例 3
脂肪来源细胞的分离与培养
在无菌条件下取吸脂术后废弃的人肌腱组织， 转移到培养瓶中， 用生理盐水 反复冲洗， 加入等体积的 0.075%的 I型胶原酶（购自 Worthington, Freehold, NJ, USA), 置于 37°C恒温摇床消化， 分别于 1小时后， 300g离心 10分钟， 获得高密度 的细胞沉淀物， 弃上清和漂浮的脂肪组织， 细胞振匀， 计数后以 1X107盘密度（培 养皿直径 100mm)培养细胞， 培养液为 DMEM (购自 Gibco, Gland, Island, NY, USA) 含 10%胎牛血清， 置于 37°C、 5%C02、 100%饱和湿度的条件下培养， 24小时后首次 换液， 用 PBS反复冲洗去除漂浮的细胞， 加入培养液， 待细胞基本长满培养皿底时 进行传代（图 3)。 细胞传至所需代次， 经 0.25%胰酶消化收集细胞用于实验。
脂肪来源细胞的鉴定
将原代脂肪来源细胞培养于细胞爬片上， 至 70%汇合时， 4%多聚甲醛固定 15 分钟， 加入各类待测表面抗原的抗体， 37°C孵育 1 小时， PBS冲洗， 加入 FITC连 接的二抗 （DAK0， Carpinteria , U. S. A) ， 37°C孵育 30分钟， PBS再次冲洗， 应 用 Heochest33258 (购自 Sigma公司 （美国） ） 或 Propidium Iodide (购自 Sigma 公司 （美国） ） 进行核衬染， 激光共聚焦显微镜下观察细胞表面抗原的表达。 脂肪来源细胞的多向分化潜能
成软骨诱导
第三代细胞采用微团块培养法进行成软骨诱导， 诱导因子终浓度为 TGF-β Ι (购自 R&D公司 （美国） ） 10ng/mL， IGF (购自 R&D公司 （美国） ） 100ng/mL， Dex (购自 Sigma公司 （美国） ） 0.1 ymOl/L， 转铁蛋白 （购自 Sigma公司 （美国） ） 6.25 μ g/mL。
成脂肪诱导
第三代细胞培养 7天时进行脂肪诱导，诱导液成分为基础培养液和 0.5mmol/L IBMX (购自 Sigma公司 （美国） ) 、 1 μ mol/L Dex、 10 μ mol/L insulin (购自 Sigma 公司 （美国） ） 、 200 μ mol/L indomethacin (购自 Sigma公司 （美国） ） ， 倒置 相差显微镜下观察细胞形态的变化， 诱导 14天进行相应的检测。
成骨诱导
第三代细胞培养 7天时进行成骨诱导，诱导液成分为基础培养液加入 2.16g/L β _磷酸甘油、 lOnmol/L维生素 D3。 诱导 20天， 对矿化结节进行成骨的相关特殊 染色。 实施例 4
生物材料支架的制备
将材料纤维预塑形成束状 （图 9) ， 并固定在 "U" 形弹簧上给予支架持续的 静态张力 （图 10) ， 再在 75%乙醇中浸泡消毒 1小时后用 PBS冲洗 3— 5遍， 紫外 灯消毒、 风干后待用。 实施例 5
组织工程肌腱的体外构建
皮肤成纤维细胞和 /或脂肪来源细胞传至第 2〜4代，经 0.25%胰蛋白酶消化收 集细胞， 制成 2.0X107ml的细胞悬液。 将细胞悬液接种到预制好的 PGA纤维支架 上， 先将细胞一材料复合物在二氧化碳培养箱内放置 4小时， 然后加入含 10%胎牛 血清的 DMEM培养液约 30ml，继续置于培养箱内培养。以后每二天更换培养液一次， 以保证细胞营养。 大约 2周后， 细胞分泌足够的细胞外基质， 再转移到牵拉式肌腱 生物反应器的细胞培养室内进行动态牵拉培养， 培养一定时期 （较佳的为 3〜10 周）后形成能够用于肌腱缺损修复的组织工程肌腱移植物。生物反应器中加载在种 子细胞一生物材料复合物上的牵引力为 2— 20N， 牵拉频率为每日牵拉 1〜12小时， 每次牵拉持续 2〜30秒， 间隔 5〜60秒， 位移为移植物长度的 5〜30 %。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领 域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权 利要求书所限定的范围。

Claims

权 利 要 求
1.一种组织工程化肌腱移植物， 其特征在于， 它包括：
(a)药学上可接受的生物可降解材料； 和
(b)种子细胞， 所述的种子细胞接种于所述的生物可降解材料， 且种子细胞 选自下组： （i)成纤维细胞， （ii)脂肪来源细胞， 或（iii) 1:10000— 10000:1 的成纤维细胞和脂肪来源细胞的混合物。
2.如权利要求 1所述的移植物， 其特征在于， 最大张力为 10— 80N。
3.如权利要求 1所述的移植物， 其特征在于， 所述的种子细胞是成纤维细胞 或成纤维细胞和脂肪来源细胞的混合物。
4.如权利要求 1所述的移植物， 其特征在于， 所述的种子细胞的含量为 IX
105个细胞 /ml-5X 108个细胞 /ml。
5.如权利要求 1所述的移植物， 其特征在于， 所述的生物可降解材料为条索 状。
6.如权利要求 1所述的移植物， 其特征在于， 所述的成纤维细胞和脂肪来源 细胞来源于自体或同种异体。
7.如权利要求 1所述的移植物， 其特征在于， 所述的药学上可接受的生物可 降解材料选自下组： 聚乳酸、 聚羟基乙酸、 聚羟基丁酸、 聚酸酐、 聚偶磷氮、 聚 氨基酸、 假聚氨基酸、 聚原酸酯、 聚酯尿烷、 聚碳酸酯、 聚乙二醇、 聚环氧乙烷、 聚对二氧六环酮、 胶原、 明胶、 糖氨聚糖、 壳聚糖、 甲壳素、 海藻酸盐、 藻酸钙 凝胶、 脱细胞基质， 及其各种类型和比列的混合物。
8.一种制备如权利要求 1所述的组织工程化肌腱移植物的方法，其特征在于， 包括步骤：
将种子细胞与药学上可接受的生物可降解材料混合， 得到种子细胞一生物材 料复合物， 其中所述的种子细胞接种于所述的生物可降解材料， 且种子细胞选自 下组： （i) 成纤维细胞， （ii) 脂肪来源细胞， 或 （iii) 1:10000〜10000:1的 成纤维细胞和脂肪来源细胞的混合物；
9.如权利要求 8所述的方法， 其特征在于， 它还包括步骤：
将种子细胞一生物材料复合物在生物反应器中培养得到如权利要求 1所述的 组织工程化肌腱移植物。
10.—种如权利要求 1 所述的组织工程化肌腱移植物的用途， 其特征在于， 它可用于制备修复肌腱缺损的移植物。
PCT/CN2008/070566 2007-06-29 2008-03-24 Tendon d'ingénierie tissulaire et procédés de construction in vitro de celui-ci WO2009003375A1 (fr)

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