WO2008071074A1 - Utilisation de cellules souches mésenchymateuses, et procédé pour isoler des cellules couches dans des tissus humains et les conserver - Google Patents

Utilisation de cellules souches mésenchymateuses, et procédé pour isoler des cellules couches dans des tissus humains et les conserver Download PDF

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WO2008071074A1
WO2008071074A1 PCT/CN2007/003505 CN2007003505W WO2008071074A1 WO 2008071074 A1 WO2008071074 A1 WO 2008071074A1 CN 2007003505 W CN2007003505 W CN 2007003505W WO 2008071074 A1 WO2008071074 A1 WO 2008071074A1
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stem cells
tissue
mesenchymal stem
human
cells
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PCT/CN2007/003505
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Chinese (zh)
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Songling Wang
Songtao Shi
Yan Xu
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Songling Wang
Songtao Shi
Yan Xu
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    • 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
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • 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
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates
    • C12N2533/18—Calcium salts, e.g. apatite, Mineral components from bones, teeth, shells
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • mesenchymal stem cells and methods for isolating and preserving stem cells in human tissues.
  • the present invention relates to the use of mesenchymal stem cells, particularly for functional repair or regeneration of mammalian tissues such as human damaged tissue or defective tissue and beauty.
  • the present invention also relates to a method of preserving stem cells, particularly a method of preserving dental pulp stem cells. Background technique
  • MSCs Mesenchymal stem cells
  • Bone marrow mesenchymal stem cells are mesenchymal stem cells with multi-differentiation ability, which can differentiate into various cells such as osteoblasts, chondrocytes, adipocytes, cardiomyocytes, myoblasts and nerve cells. These cells were originally distinguished by a cell population of cells formed in vitro by fibroblast morphology (CFU-F population forming unit-matrix). Each group appears as a cell line in which one cell begins to proliferate. Although bone marrow mesenchymal stem cells have the ability to differentiate into a variety of cells, they usually tend to form bone tissue. For example, bone marrow mesenchymal stem cells can continue to form bone/medullary after being transplanted into the immunodeficient mouse.
  • Organoid structure Another surprising feature is that in xenogeneic transplantation, bone marrow mesenchymal stem cells activate and support the hematopoietic component during osteogenesis. This manifestation of mesenchymal stem cells with various differentiation potentials strives to establish the microenvironment from which they originate. Mesenchymal stem cells have also been successfully used in fractures, aplastic anemia, and in the treatment of human type IV acute graft-versus-host disease by modulating immune cell responses. In addition, bone marrow mesenchymal stem cells are also used to generate adipose tissue for filling and reconstitution of soft tissues.
  • Periodontal disease is the leading cause of tooth loss in adults.
  • Treatment of periodontal disease is a difficult problem in clinical work.
  • Conventional regenerative treatments include guided tissue regeneration (GTR), topical application of enamel derivatives (EMD), or various growth factors that can cause tissue regeneration in the periodontal portion. These treatments have varying effects, mainly related to the missing shape and the amount of healthy normal periodontal ligament. So far, there is no treatment that can achieve functional regeneration of periodontal tissue.
  • mesenchymal stem cells such as bone marrow mesenchymal stem cells or periodontal (ligament) mesenchymal stem cells (PDLSCs) have shown unexpectedly good effects in functionally repairing human damaged tissues or defective tissues and cosmetics.
  • bone marrow mesenchymal stem cells repair facial damage or defects in mammals such as humans, periodontal mesenchyme repair, and the above repairs are not only tissue shape repair, but also recovery or repair of la-texture regeneration and normal function.
  • the present inventors have further found in the research that the special treatment of stem cells in human tissues can advantageously retain the activity of stem cells in human tissues, thereby providing a possibility for the repair of tissue damage in humans in the future.
  • the present invention has been completed based on the above findings.
  • a first aspect of the invention relates to the use of mesenchymal stem cells for the preparation of a product for functional repair or regeneration of a mammalian, e.g., human, damaged tissue or defective tissue or cosmetic.
  • Another aspect of the invention relates to a method of functionally repairing or regenerating a mammalian, such as a human damaged tissue or defective tissue or cosmetic, comprising administering a functionally repairing effective amount of mesenchymal stem cells to a mammal in need of functional repair of damaged or defective tissue.
  • Animals such as humans or humans who need beauty.
  • the invention also relates to a functional repair or regeneration mammalian such as a human injury group A composition of woven or defective tissue comprising mesenchymal stem cells and hydroxyapatite/calcium triphosphate or gelatin sponge.
  • the invention further relates to compositions for use in cosmetics comprising mesenchymal stem cells and hydroxyapatite/calcium triphosphate or gelatin sponge.
  • the invention also relates to a method for isolating and preserving stem cells in human tissues, which comprises chopping human tissue containing stem cells, and then digesting in an enzyme solution consisting of type I collagenase and neutral protease at 37 t; (PH6-9) 10-60 min; single cell suspension was filtered and the resulting single cells were washed with Ca 2+ and Mg 2+ -free fluorescent buffer (PBS-) and then at 4.
  • C is mixed with 90% fetal calf serum (FBS) and 10% dimercaptosulfoxide, placed at low temperature, and then refrigerated in liquid nitrogen.
  • the term "functional repair or regeneration” means that a damaged tissue or a defective tissue of a mammal such as a human is treated by the mesenchymal stem cells of the present invention, and can not only return or restore or improve to a normal tissue shape but also have normal physiological functions of normal tissues. . If the damaged periodontal tissue is treated by the periodontal mesenchymal stem cells of the present invention, it can be regenerated into normal periodontal tissue, thereby regenerating normal teeth.
  • meenchymal stem cells is exemplified by bone marrow mesenchymal stem cells and periodontal ligament (mesenchymal) stem cells (PDLSCs).
  • PDLSCs bone marrow mesenchymal stem cells and periodontal ligament (mesenchymal) stem cells (PDLSCs).
  • the PDLSCs are preferably from humans under the age of 20, and the PDLSCs are freshly removed and frozen and reused. The same function, and there is no limit to the freezing time.
  • injured or defective tissue is, for example, a tissue damage or defect caused by disease, genetic or external force such as trauma.
  • cosmetic is used, for example, to modify or reshape the contours of a mammal, such as a human face, and to repair or regenerate the aging of a mammal, such as a human skin, such as the removal of facial wrinkles or freckles, and is generally understood by those skilled in the art. Beauty category.
  • human tissue generally refers to soft tissue, which generally refers to the removal of bone, teeth. All tissues of the quality and blood components have no hard tissue.
  • low temperature generally means below O, for example, -80 °C.
  • Figure 1 is: Human bone marrow msc-mediated reconstruction of the mouse face
  • C H&E staining showed a large amount of active bone tissue (B) and hematopoietic bone marrow (BM) produced by bone marrow MSC grafts.
  • B active bone tissue
  • BM hematopoietic bone marrow
  • the black arrow shows the height of the regenerated bone.
  • Figure 2 is a description of the MSC regenerated bone in the maxillofacial region
  • CF Immunohistochemical analysis with anti-ALP (C), type III collagen (CIII, D), MEPE (E) antibody revealed that osteogenesis cells (black arrows) or osteoblasts resemble newly formed bone surfaces, Or similar to bone cells in newly formed bone. Immunohistochemistry IgG staining was negative (F)
  • MSC grafts contain well-differentiated bone and bone marrow components, showing typical hematopoietic niche structures, including osteoblasts (empty arrows) and hematopoietic cells (BM). H&E staining
  • FIG. 3 GFP-positive bone marrow cells homing into the bone marrow component of MSC (A, B) GFP bone marrow was injected into the MSC/HA transplant recipient mice via the tail vein. Immunohistochemical analysis with anti-GFP monoclonal antibody after 2 months of injection showed GFP-positive bone marrow ( ⁇ homing into the bone marrow of MSC graft ( )
  • C Cell flow analysis of the heaviest cells of MSC grafts or untransplanted long bones, GFP-positive cells and hematopoietic cell markers (upper right square) CD45, CD9, CDllb co-expressed in bone marrow from MSC grafts . (lower layer). Bone marrow cells (upper layer) taken from the long bones of the transplanted rat were used as a negative control.
  • C, D 3D reconstruction showing the skeletal appearance of MSC/HA grafts (empty arrow) (C) and pigs without MSC transplantation (D)
  • E, F Coronal CT plain scan image.
  • the MSC graft (white arrow) is tightly bound to the skull (yellow arrow) (E). There was no bone connection between the graft and the skull in the control group (F).
  • Figure 5 is a description of pig MSC regenerated bone
  • A-D Electron microscopy analysis of MSC/HA and HA/TCP grafts. Numerous micropores (empty arrows) are present on the broken surface, including the inner (A) and outer (B) sides of the HA/TCP. After 8 weeks of MSC transplantation, these spaces became smaller compared to HA/TCP grafts, either inside (C) or outside (D).
  • Figure 6 is a view of the reduction of wrinkles caused by human MSCs in rats.
  • bone marrow MSC/gelatin sponge does not produce collagen fibers, either after 2 weeks of transplantation (C) or after 4 weeks (D). Although some cellular components appeared after two weeks of transplantation, they could not form collagen fibers (C). After 4 weeks of transplantation, only a limited number of cells appeared. (D)
  • E-H In situ hybridization was performed on MSC grafts using human alu. and mouse specific pfl. A large number of human alu. positive (black spots) cells appeared (P) after PDLSCs/gelatin sponges were transferred to plants, and pfl-positive (black spots) cells were detected only outside the graft (G). In situ hybridization with sense mRNA showed negative staining for both alu and pfl (H).
  • the (IL) single-column PDLSCs act similarly to the mixed colony PDLSCs, both producing compressed collagen fibers, as shown by H&E staining (1, black arrow in K) and polarized light in the same field, U, L White arrow in the middle).
  • Figure 8 is:
  • (B) PDLSCs can be extracted to produce similar collagen, but longer.
  • Histology shows regeneration of new bone (C) and periodontal tissue (H), as well as newly formed cementum (F)
  • CT showed significant bone damage before transplantation (J, L). After three months of transplantation, bone tissue in PDLSCs-HA/TCP group was completely regenerated (K), and there was almost no regeneration in the HA/TCP group (M).
  • Figure 9 shows: Compared with the other two groups, the PDLSCs experimental group showed significant bone regeneration after 3 months of transplantation.
  • the bone marrow mesenchymal stem cells or periodontal ligament stem cells of the present invention are usually used in combination with hydroxyapatite/calcium triphosphate (HA/TCP) or gelatin sponge.
  • HA/TCP hydroxyapatite/calcium triphosphate
  • FIG 10 is a view: The cryopreserved pulp mononuclear cells contain dental pulp stem cells (A, B). A single colony, cell colony forming unit (CFU-f) (A), is formed on the medium. In terms of the ability to form (CFU-f), compared with normal pulp mononuclear cells, there was a significant decrease in the number of pulp mononuclear cells after refrigeration (CE).
  • CE pulp mononuclear cells after refrigeration
  • DPSCcryo monocytes dental pulp stem cells
  • STR0- 1 MUC18, CD105, and CD73 positive (F.), negative to CD34, I gM and 1 8 0 1 is Used as a negative control.
  • Figure 11 shows: DPSCcryo has similar differentiation ability to PDSC.
  • ALP Alkaline phosphatase
  • B L-ascorbic acid-2- Magnesium phosphate, dexamethasone, and inorganic phosphate were stained with alizarin red for four weeks and showed the same level of mineral nodule formation.
  • C, D DPSCcryo is similar to normal PDSC, using real-time PCR (C) Detection of bone viscous protein (ON) and bone The nuclease (0CN), measured by the Wes tern b lot method, has RUNX2, ALP, and OCN (D). G3PDH and - used as internal control.
  • E, F when cultured containing 0.5 mM phosphodiesterase Inhibitor (IBMX), 0.5 mM hydrocortisone, 60 mM armor. After 5 weeks in a new fat-inducing environment, similar to normal PDSC, a lipid-positive lipid group (E) can be formed.
  • DPSCcryo can form dentin in vivo. After 8 weeks of transplantation, DPSCcryo can differentiate into dentate cells (empty arrows), the cells It is the cause of the formation of a dentin-like structure on the surface of the hydroxyapatite carrier (HE staining) (A). DPSCcryo produces the same dentin structure (A) and a similar amount of dentin (B) as the DPSC. (0) In immunocytochemical staining, the dentin structures produced by DPSCcryo and DPSC are positive for human-specific mitochondria and DSP antibodies.
  • the third molars (n 18) from 16 normal adults (18-20 years old).
  • the periodontal ligament was carefully separated from the root surface of the tooth and then placed in 3 mg/ml type I collagenase.
  • the removed MSC graft was dewaxed and then coated with primary antibody (1:200-1:500 dilution) for one hour. Immunohistochemistry experiments were performed according to the instructions using the Zymed SuperPicTure Polymer Assay Kit (Zymed nvitrogen). Pig model
  • HA/TCP-MSCs were placed in the experimental group of 3 pig periosteum for 8 weeks, and HA/TCP was placed in the body of the control group.
  • the MSC graft sample was cut into approximately 1 leg pieces and fixed with 2% polyfurfural and 2.5% glutaraldehyde for 2 hours. Rinse with sodium dicitrate and fix in 1% osmium tetroxide. Dehydrated with graded alcohol and then incubated in isoamyl acetate. After gold plating, the samples were tested under a Toshiba S-520 electron microscope. In situ hybridization
  • the starters include: human alu, sense, 5'-tggctcacgcctgtaatcc-3' (base number: 90-108), ant isense, 5'-TTTTTTGAGACGGAGTCTCGC-3' (BASE NUMBER: 344-364, Genbank accession number: AC004024) ; and mouse pfl, sense, 5'-CCGGGCAGTG GTGGCGCATGCCTTTAAATCCC-3' (base number: 170-201), ant isense, 5'-GTTTGGTTTTTGAGCAGGGTTCTCTGTGTAGC-3' (base number: 275-306, Genbank access ion number: X78319).
  • PCR polymerase chain reaction
  • 1 PCR buffer Perkin Elmer, Fos ter City, CA
  • 0.1 mM dATP 0.1 mM dCTP
  • 0.1 mM dGTP 0. 065 mM dTTP
  • 0. 035 mM digoxigenin-l l-dUTP 10 pmol of a specific initiator
  • 100 ng of human chromosomal DNA as a template.
  • Unstained samples were deparaffinized and hybridized with the digoxigenin-labeled a lu or pf 1 probe by mRNA loca tor-Hyb Ki t (Ambion, Inc., Aus t in ⁇ ).
  • the presence of alu or pf1 in the tissue can be detected by NBC/BCIP solution (RocheDiagnos tic/Boehringer Mannheim Corp) after an immunological reaction with an anti-digoxigenin ALP binding fragment (RocheDiagnos tic/Boehringer Mannheim Corp). Bone marrow cells homing
  • Rats transplanted with MSC graft for 8 weeks were intravenously injected with cyclophosphamide (Sigma) diluted in phosphate buffered saline (PBS) at 64 mg/kg once daily for 4 days. Then, the transgenic mouse long bone-expanded GFP (sGFP)-producing bone marrow cells (1.57 7 all nuclear cells/body) were injected into the cyclophosphamide-injected mouse through the tail vein. Eight weeks after the injection, eGFP-positive homing bone marrow cells in the MSC graft can be detected by immunohistochemistry and fluorescence activated cell sorting. Fluorescence activated cell sorting (FACS) analysis
  • HA/TCP grafts only resulted in a limited amount of induced bone formation between the HA/TCP particles and the calvarial contact surface (Fig. 1D). Further studies revealed that the newly formed bone was connected to the rat skull by connective tissue and directly bound to HA/TCP (Fig. 2B). This also confirmed that osteoblasts in MSC secrete osteogenic factors, including ALP, type III collagen, and MEPE (Fig. 2C-2F). MSCs play a reliable role in the osteogenesis of the maxillofacial region.
  • Piglets were used as a transplant model, and homologous bone marrow MSCs were placed on HA/TCP for transplantation into the maxillofacial region. Similar to experiments in rats, bone marrow MSCs were found to alter the appearance of the maxillofacial region (Fig. 4A, 4B). Imaging studies revealed a osseointegration of the MSC graft with the frontal bone of the recipient (Fig. 4C-4H), which provides evidence for the application of this technique to orthopedics. Animals transplanted with autologous bone marrow MSCs showed massive bone regeneration compared to the single-transplant HA/TCP animal model (Fig. 5). According to electron microscopy and histology analysis, pig autologous MSCs caused high-quality bone tissue regeneration on MSC grafts of different surfaces (Fig. 5).
  • Example 2 Example 2
  • the PDLSCs were transplanted into the skin of the rats to reduce facial wrinkles (Fig. 6).
  • HA/TCP grafts carrying PDLSCs are placed under the skin, they form collagen fibers and cementum in the body.
  • PDLSCs were implanted into a collagen-based gelatin sponge and transplanted into the body to produce a large amount of collagen fibers (Fig. 6A, 6B).
  • Fig. 6M, 6m, 6N, 6n Prior to this, collagen gels and stabilized hyaluronic acid have been used for non-surgical cosmetic purposes, and fibroblasts of the dermis are used as a filling for soft tissue.
  • MSCs Human mesenchymal stem cells are thought to be primitive cells with multiple directional differentiation capabilities that can differentiate into osteoblasts, chondrocytes, fat cells, muscles and nerves (3, 9, 20, 30-32). MSCs have now been used to treat severe bone damage that cannot be healed naturally (3, 20, 30, 32-34). Histological analysis showed that MSCs-mediated bone regeneration was similar in the maxillofacial region to the subcutaneous site previously studied (18, 20). Studies on ectopic tissue regeneration from previous MSCs have shown that it has the ability to be used in the reconstruction of maxillofacial bones, accompanied by maintenance of HSC niches. These results show that MSCs will become an important material for maxillofacial tissue reconstruction.
  • MSC grafts have the ability to regenerate functional organ-like structures that work in conjunction with the recipient body.
  • the advantage of this tissue regeneration is that it provides a long-term tissue re-establishment by developing a balance between the hematopoietic microenvironment between the graft and the recipient host.
  • bone formation was also found in the control group in which HA/TCP particles were transplanted, the number of bone formation was small, and there was no production of hematopoietic components in the control group.
  • Transplanted PDLSCs produce a large amount of collagen fibers that are similar in structure to the original PDL.
  • transplanted MSCs and gingival fibroblasts do not produce any tissue, probably because they do not produce the ideal niche to maintain viability.
  • Different carriers have a great influence on the production of collagen fibers.
  • gelatin sponges allow PDLSCs to produce collagen fibers in the body without producing inorganic-containing tissue components, while HA/TCP-PDLSCs grafts produce both collagen fibers and cementum.
  • the mechanism by which different vectors cause PDLSCs to produce different products is still not well understood, and may be due to the ineffective difference in biocompatibility of the vector or the ability to induce differentiation of the MSC. This feature provides a unique opportunity for PDLSCs to improve facial appearance.
  • Example 1 demonstrates that MSCs can be used for integers to improve facial appearance.
  • Experimental Example 2 shows that PDLSCs can be used for human beauty, such as wrinkling of the face.
  • Example 3 Method for repairing and regenerating periodontal tissue damage caused by periodontal disease by PDLSCs
  • the canine teeth of the mini-pigs were removed, and the ligaments around the middle third of the roots were separated, and the ligaments were placed in the solution for 37 hours.
  • the digestive juices included 3 mg/ml type I zymogen (Wor thing ton biochemical company) , Freeho ld, NJ ) and 4mg/ml dis pa se (Roche, Mannhe im, Germany), after 70um filter (Fa l con, BD Labware, Frankl in Lakes, NJ, USA) . Filtration to obtain a single cell suspension. PDLSCs from different individuals were cultured separately.
  • a single cell suspension was placed in a 10 cm tissue plate (Cos tar, Cambr idge, MA) planted in a-MEM medium (GIBCO/Invitrogen, Carlsbad, CA) from 15% fetal Bovine serum (Equi tech-Bio Inc, Kerrvi l le, TX) l OOuM L-ascorbic acid-2-phosphate (WAK0, Tokyo, Japan), 2 mM L-glutamine, 100 units/ml penicillin and 100 ug/ml chain
  • the mycin Biosource/Invi trogen
  • the efficiency of colony formation was evaluated on day 14.
  • a colony of more than 50 cells was identified as a colony.
  • Stage 3 PDLSCs were placed in the induction medium (0.5 mM 3-isobutyl-1-mercaptopurine (IBMX), 0.5 M hydrocortisone and 60 M armor). Oil red 0 after 4 weeks ( Sigma) is used to detect fat cells. Immunohistochemical staining
  • PDLSCs were placed in 24-well cell culture slides (2 X 10 4 cells/well, NUNC, Napervi le, IL, USA) for secondary culture. The cells were first fixed in 4% paraformaldehyde for 15 minutes and then blocked and coated with anti-Stro-1 (R&D, 1:200-1:500 dilution) for one hour, according to the manufacturer's instructions. Then put any IgM secondary antibody in the sheep for 45 minutes. Production of animal models of periodontitis
  • the alveolar bone in the middle of the maxillary or mandibular first molar was destroyed, and the i-ligament of the root was sutured with a 4th thread.
  • the strap was removed after 10 days.
  • the size of the defect is 3mm wide, 7 inches long and 5mm deep.
  • a bone valve appears during the procedure and the alveolar bone is removed.
  • a concave cover is placed between the crown of the alveolar bone and the lowermost portion of the damaged portion.
  • the animals after the surgery are trained for 30 days to maintain the periodontal condition and then the next operation can be performed.
  • PKI Plaque Index
  • SBI sulcus bleeding index
  • PD periodontal probing depth
  • CAL clinical attachment loss
  • the histological evaluation of the defect tissue was performed at different time points after operation.
  • the sample was first fixed in 4% formalin, and then a portion of the sample was placed in 10% edetate buffer to remove lime and finally coated with paraffin.
  • the sample was dewaxed and stained with H&E. Another part of the sample was embedded in plastic without lime treatment (Dona th and Breuner, 1982).
  • the unstained portion of the sample was first detected by fluorescence microscopy, and then the sample was stained with indoline blue and observed under a light microscope. Bone mineral density and regeneration at different time periods were analyzed by X-ray scanning and CT (S i emens Company, Germany). Scanning thickness is 0. 75mm.
  • Statistical analysis The mean of the clinical data was statistically tested. The clinical data of the three groups were compared with each other and the pre- and post-operative data were compared by the F test. The p-value was less than 0.05.
  • the alveolar bone density at the affected site was significantly reduced 6 months after surgery. (Fig. 7F). Significant bone damage was observed in both the C T 3D reconstruction and the crown scan (Fig. 7G, H). This shows that surgical removal of part of the alveolar bone while ligating the surrounding portion can produce a stable periodontitis model.
  • transplanting cells to the periodontal disease area can regenerate the cementum, periodontal ligament, and alveolar bone.
  • the ability to regenerate from different cells is very different. It is not only related to the transplant site, but also related to the active differentiation of the transplanted cells.
  • Previous studies have shown that early transplantation of mesenchymal cells with diseased periodontal can significantly reduce epithelial to root hyperplasia and promote stable structural formation.
  • the goal of periodontal disease treatment is to activate the remaining periodontal stem cells to produce new cementum and bone components.
  • various inflammatory factors such as IL-1, TNF-a, and MMP destroy periodontal support tissues and alter the function of residual periodontal stem cells.
  • stem cells that can differentiate into bone and cementum surface are reduced. Therefore, direct transfer of stem cells into periodontal tissue should be a good treatment.
  • Previous experiments have shown that stem cells, either from fresh or frozen periodontal ligaments, have the ability to differentiate into cementum/osteoblasts in vitro and form cementum/periodontal ligament-like tissue in vivo.
  • we successfully isolated PDLSCs from minipigs and demonstrated that these cultured cells have the same characteristics as human cells. In 170 culture from single cells 105 out of the low density population of single colonies, they behave as mesenchymal stem cell markers STR0-1 positive (5.6% positive). These ones PDLSCs can also produce collagen and differentiate into adipocytes in vitro.
  • the maxillofacial region of miniature pigs has many similarities in terms of anatomy, physiology, disease, and disease. Gingivitis is a common disease in small pigs after 6 months. More severe periodontitis can occur in pigs older than 16 months. There are various methods for making periodontal disease models in pigs, such as ligation, bacteria, bone damage, and elastic metal shaping. Our animal model was made with silk thread for 10 days of ligation while bone damage occurred. Observing this method within 6 months after surgery, this method can significantly increase PD and CAL. Within one month after surgery, PD and CAL increased significantly, and repair began one month later, but did not return to normal levels.
  • PDSCs dental pulp stem cells
  • the method of tooth extraction was performed according to the University of Southern California guidelines.
  • the method of separating dental pulp stem cells as previously described (Gronthos et al., 2000) 0 is mainly to separate the molars along the cementum enamel junction with a sterile forceps.
  • the pulp tissue is separated from the crown and the root cavity.
  • the sample was minced and digested in a 37 ° C enzyme solution for 30 minutes.
  • the enzyme solution consisted of 3 mg/mL type I collagenase (Worthington Biothech, Freehold, NJ) and 4 mg/mL neutral protein drunk (Roche Diagnotic/Boer inger Mannheim Corp., Indianapol is, IN).
  • Single cell suspensions were obtained by filtration through a 70- ⁇ filter (Falcon, BD Bioscience, Franklin Lakes, NJ).
  • the resulting monocytes are generally used for pre-culture (Gronthos et al., 2000) and the other half is refrigerated.
  • mononuclear cells were washed 3 times with Ca 2+ and Mg 2+ -free fluorescent buffer (PBS-) and applied at 4 ° C with 90% fetal calf serum (FBS) (Equitech- Bio Inc., Kerrville, TX) mixed with 10% dimethyl sulfoxide (Sigma, St Louis, MO) (operating on ice).
  • FBS fetal calf serum
  • the cells were stored at -80 ° C overnight and refrigerated with liquid nitrogen. After storage for 6 months in cold storage, it was at 37. It melts rapidly in the C state and is used for the culture of DPSC.
  • Monocytes were seeded in 100 mm cultures (Coaster, Cambridge, MA) as ⁇ 3 and unattached cells were removed 3 hours later.
  • the attached cells were supplied with nutrients from ⁇ - ⁇ (Invitorgen Co. Grand Island, NY), including 15% FBS, 100-mM magnesium monophosphate (Wako Purre Chemicals, Osaka Japan), 2 mM glutamine, 100 U/ Ml penicillin I 100 g/ml streptomycin (Biosource, Rockville, MD).
  • the resulting cell colonies are the first stage of PDSCs.

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Abstract

L'invention concerne un procédé pour isoler des cellules souches dans des tissus humains et conserver ces cellules souches. Des cellules souches mésenchymateuses sont utilisées de manière spécifique pour la restauration et la régénération fonctionnelles de tissus blessés ou défectueux chez les mammifères tels que les êtres humains, ainsi qu'en coiffure.
PCT/CN2007/003505 2006-12-13 2007-12-10 Utilisation de cellules souches mésenchymateuses, et procédé pour isoler des cellules couches dans des tissus humains et les conserver WO2008071074A1 (fr)

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CN200610165839 2006-12-13
CN200610165839.6 2006-12-13

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WO2008071074A1 true WO2008071074A1 (fr) 2008-06-19

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110800732A (zh) * 2019-10-10 2020-02-18 湖南源品细胞生物科技有限公司 一种脐带间充质干细胞贮藏液
CN111094550A (zh) * 2017-09-08 2020-05-01 株式会社大塚制药工厂 来源于幼猪的干细胞及其制备方法

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CN1422672A (zh) * 2002-11-21 2003-06-11 上海第二医科大学附属第九人民医院 可吸收煅烧骨的制备方法
CN1511593A (zh) * 2002-12-30 2004-07-14 中国人民解放军军事医学科学院放射医 一种含有人骨髓间充质干细胞的人工皮肤及其构建方法
CN1565644A (zh) * 2003-06-27 2005-01-19 中国人民解放军军事医学科学院基础医学研究所 一种组织工程化骨的制造方法
CN1590537A (zh) * 2003-09-02 2005-03-09 中国人民解放军第四军医大学口腔医学院 外胚间充质干细胞的分离和培养方法

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CN1422672A (zh) * 2002-11-21 2003-06-11 上海第二医科大学附属第九人民医院 可吸收煅烧骨的制备方法
CN1511593A (zh) * 2002-12-30 2004-07-14 中国人民解放军军事医学科学院放射医 一种含有人骨髓间充质干细胞的人工皮肤及其构建方法
CN1565644A (zh) * 2003-06-27 2005-01-19 中国人民解放军军事医学科学院基础医学研究所 一种组织工程化骨的制造方法
CN1590537A (zh) * 2003-09-02 2005-03-09 中国人民解放军第四军医大学口腔医学院 外胚间充质干细胞的分离和培养方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111094550A (zh) * 2017-09-08 2020-05-01 株式会社大塚制药工厂 来源于幼猪的干细胞及其制备方法
CN110800732A (zh) * 2019-10-10 2020-02-18 湖南源品细胞生物科技有限公司 一种脐带间充质干细胞贮藏液

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