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Oct 2020
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Mouse Periosteal Cell Culture, in vitro Differentiation, and in vivo Transplantation in Tibial Fractures
小鼠骨膜细胞培养、体外分化及胫骨骨折的体内移植   

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Abstract

The periosteum covering the outer surface of bone contains skeletal stem/progenitor cells that can efficiently form cartilage and bone during bone repair. Several methods have been described to isolate periosteal cells based on bone scraping and/or enzymatic digestion. Here, we describe an explant culture method to isolate periosteum-derived stem/progenitor cells for subsequent in vitro and in vivo analyses. Periosteal cells (PCs) isolated using this protocol express mesenchymal markers, can be expanded in vitro, and exhibit high regenerative potential after in vivo transplantation at a fracture site, suggesting that this protocol can be employed for PC production to use in new cell-based therapies.

Keywords: Periosteum (骨膜), Bone regeneration (骨再生), Skeletal stem/progenitor cell (骨骼干细胞/祖细胞), In vivo cell transplantation (体内细胞移植), In vitro differentiation (在体外分化)

Background

Bone regeneration is a highly efficient process. After bone fracture, skeletal stem/progenitor cells are activated and differentiate into chondrocytes and osteoblasts that form cartilage and bone to consolidate the fracture. Skeletal stem/progenitor cells originate from the bone itself including bone-marrow and periosteum, as well as from adjacent soft tissue. The diverse origins of skeletal stem/progenitor cells during bone regeneration suggest that these cells can be obtained from various sources for stem cell therapies. Due to their accessibility, bone marrow stromal cells (BMSCs) are the most studied (Arthur and Gronthos, 2020); however, their variable osteogenic potential highlights the need for new sources of cells capable of contributing efficiently to the repair process. Recent studies have revealed the role of the periosteum as an essential source of stem/progenitor cells during bone regeneration (Debnath et al., 2018; Duchamp de Lageneste et al., 2018; Ortinau et al., 2019). When transplanted to a bone fracture site, periosteal cells (PCs) display a higher regenerative potential than BMSCs and have the ability to correct a bone repair failure (Duchamp de Lageneste et al., 2018; Julien et al., 2020). Isolating mouse PCs is challenging since the periosteum is a very thin layer of tissue on the outer surface of bone. Several methods have been previously described to isolate PCs in mice, relying on enzymatic digestion and periosteum scraping or peeling (Brownlow et al., 2000; Arnsdorf et al., 2009; Wang et al., 2010; Chang and Knothe Tate, 2012; van Gastel et al., 2012). Here, we describe a method to isolate PCs based on cell migration from bone explants without digestion or periosteum separation from the bone (Figure 1). We developed this method to analyze PC properties for direct comparison with BMSCs that are usually isolated by direct bone marrow flushing and plating. Long bones free of skeletal muscle, epiphyses, and bone-marrow are placed in culture to allow PC migration and proliferation. PCs isolated using this protocol express mesenchymal markers (Figure 2), display in vitro adipogenic, osteogenic, and chondrogenic differentiation capacities (Figure 3), and are able to form cartilage and bone upon in vivo transplantation at the site of a tibial fracture (Figure 4). PCs therefore maintain their osteochondrogenic capacities, offering new potential perspectives for the study of PCs and their use in cell-based therapies.

Materials and Reagents

  1. Falcon® 5-ml round-bottomed polystyrene test tubes (Corning, catalog number: 352235)

  2. 40-μm cell strainer (Fisher Scientific, catalog number: 352340)

  3. Conical tubes, 15-ml and 50-ml (Falcon, catalog numbers: 352097 [15 ml] and 352070 [50 ml] or equivalent)

  4. 25 G needles (Terumo, catalog number: AN*2516R1)

  5. 1-ml syringes (Terumo, catalog number: SS+01H1)

  6. 60-mm TPP culture dishes (TPP, catalog number: 93060)

  7. 100-mm TPP culture dishes (TPP, catalog number: 93100)

  8. 6-well plates (TPP, catalog number: 009206)

  9. Greiner Bio-One Petri dishes (bacterial dish; Dutscher, catalog number: 633185)

  10. 10-ml and 25-ml pipets (Dutscher, catalog numbers: 357551 [10 ml] and 357535 [25 ml] or equivalent)

  11. Cell scrapers (TPP, catalog number: 99010 or equivalent)

  12. Kova® slides (Fisher Scientific, catalog number: 22-270141)

  13. Falcon® 5-ml round-bottomed polystyrene test tubes (Corning, catalog number: 352235)

  14. Glass slides, Superfrost Plus (Thermo Fisher, catalog number: J1800AMNZ)

  15. Coverslips (Labellians, catalog number: LCO2460M)

  16. For periosteum-derived cell culture: 4 to 8-week-old mice in the C57BL/6J background (see Note 1)

  17. Hosts for in vivo cell transplantation: 10 to 14-week-old mice in the C57BL/6J background (see Note 2)

  18. DMEM (Life Technologies, catalog number: 11966-025)

  19. Penicillin-streptomycin (P/S; Life Technologies, catalog number:15140-122)

  20. α-Modified Eagle’s Medium (α-MEM with GlutaMAX; Life Technologies, catalog number: 32561-029)

  21. Lot-selected fetal bovine serum (FBS; Life Technologies, catalog number:10270-106)

  22. Recombinant mouse basic fibroblast growth factor (bFGF; R&D, catalog number: 3139FB/CF)

  23. Trypan Blue stain (Life Technologies, catalog number: 15250-061)

  24. Trypsin-EDTA (0.25%), phenol red (Life Technologies, catalog number: 25200056)

  25. Dexamethasone (Sigma-Aldrich, catalog number: D8893)

  26. Human insulin solution (Sigma-Aldrich, catalog number: I9278)

  27. Indomethacin (Sigma-Aldrich, catalog number: I7378)

  28. 3-Isobutyl-1-methylxantine (IBMX, Sigma-Aldrich, catalog number: I5879)

  29. Oil Red O (Sigma-Aldrich, catalog number: O0625)

  30. Isopropanol (Sigma-Aldrich, catalog number: I9516)

  31. L-ascorbic acid (Sigma-Aldrich, catalog number: A8960)

  32. Beta-glycerophosphate (Sigma-Aldrich, catalog number: G9422)

  33. Alizarin Red S (Sigma-Aldrich, catalog number: A5533)

  34. DMEM high glucose (Life Technologies, catalog number: 31966-021)

  35. Sodium pyruvate (Sigma-Aldrich, catalog number: P5280)

  36. L-proline (Sigma-Aldrich, catalog number: P5607)

  37. Insulin-transferrin-sodium selenite (ITS; Sigma-Aldrich, catalog number: I1884)

  38. TGF-β1 (Sigma-Aldrich, catalog number: T7039)

  39. Alcian Blue (Sigma-Aldrich, catalog number: A5268)

  40. Glutaraldehyde (Merck, catalog number:1-04239-0250)

  41. Hydrochloric acid (Sigma-Aldrich, catalog number:H1758)

  42. Phosphate-buffered saline (PBS; Life Technologies, catalog number: 14190-094)

  43. Ethanol (Ethanol absolute ≥99.8%; VWR, catalog number: 20821.365)

  44. Hematoxylin (Sigma-Aldrich, Hematoxylin Solution, Harris Modified, catalog number: HHS32-1L)

  45. Sytox Blue dead cell stain (Life Technologies, catalog number: S34857)

  46. Tisseel matrix (Thrombin and Fibrin solution, Baxter, catalog number: 3400894252443)

  47. Sterile distilled water (Life Technologies, catalog number: 15230162)

  48. Buprenorphine (Centravet, catalog number: BUP001)

  49. Atipamezole (Centravet, catalog number: ANT201)

  50. Ketamine (Centravet, catalog number: KET205)

  51. Medetomidine (Centravet, catalog number: DOM003)

  52. Vetedine Savon (Vetoquinol, catalog number: 2608436 7/1992)

  53. Vetedine solution (Vetoquinol, catalog number: 4576889 5/1992)

  54. Paraformaldehyde 4% (PFA; Clinisciences, catalog number: sc-281692)

  55. Sucrose (VWR, catalog number: 443815S)

  56. EDTA solution 0.5 M (Euromedex, catalog number: EU0084)

  57. Tissue Freezing Medium (MMFrance, catalog number:F/TFM-C)

  58. Neo-Clear (Sigma-Aldrich, catalog number: 109843)

  59. Hematoxylin anhydrous (Sigma-Aldrich, catalog number: 109843)

  60. Iron(III) chloride, 97% (Sigma-Aldrich, catalog number: 157740)

  61. Fast Green (Sigma-Aldrich, catalog number: F7252)

  62. Safranin O (Sigma-Aldrich, catalog number: S2255)

  63. Neo-mount (Sigma-Aldrich, catalog number: 109016)

  64. Fluoromount-G Mounting medium with DAPI (Thermo Fisher, catalog number: 00-4959-52)

  65. PE-CyTM7 Rat Anti-Mouse CD31 Antibody (dilution 1/400; BD Bioscience, catalog number: 561410)

  66. PE-CyTM7 Rat Anti-Mouse CD45 Antibody (dilution 1/400; BD Bioscience, catalog number: 552848)

  67. PE-CyTM7 Rat Anti-Mouse CD11b Antibody (dilution 1/400; BD Bioscience, catalog number: 552850)

  68. BV650 Rat Anti-Mouse Ly-6A/E Antibody (dilution 1/200; BD Bioscience, catalog number: 740450)

  69. PE Hamster Anti-MouseCD29 Antibody (dilution 1/200; Miltenyi, catalog number: 130-102-994)

  70. Washing medium (see Recipes)

  71. Growth medium (see Recipes)

  72. FACS medium (see Recipes)

  73. Adipogenic medium (see Recipes)

  74. Oil Red O stock solution (see Recipes)

  75. Osteogenic medium (see Recipes)

  76. Alizarin Red S staining solution (see Recipes)

  77. Chondrogenic medium (see Recipes)

  78. 1% Alcian Blue staining solution (see Recipes)

  79. Cryoprotection solution (see Recipes)

  80. Weigert’s solution (see Recipes)

  81. Fast Green solution (see Recipes)

  82. Safranin O solution (see Recipes)

Equipment

  1. Surgical forceps (×4) (Dumont AA Forceps; FST, catalog number: 11210-20, or equivalent)

  2. Surgical scissors (×4) (Fine Scissors-ToughCut® 11 mm; FST, catalog number: 14058-11, or equivalent)

  3. BD LSRFortessa machine (Becton Dickinson)

  4. Sterile hood for cell culture

  5. Centrifuge with temperature control

  6. Water bath with temperature control

  7. CO2 incubator set at 5% CO2 and 33°C or 37°C

  8. Shaker (VWR, model: Mini nutating, 3D mixer)

  9. Drill (Dremel, catalog number: 8050-15)

  10. Drill bits (0.4 mm)

  11. W/C3 NDL Silk BL Braid sutures (Havard Apparatus, catalog number: 72-3318)

  12. Heating pad (Harvard Apparatus, catalog number: 55-7033)

  13. Mouse mower (Kerbl, catalog number: GT416)

  14. Sterile scalpels (Dutscher, catalog number: 132622)

  15. Cryostat (Leica Biosystems)

  16. MM35P blade (MMFrance, F/MM35P)

  17. Zeiss Imager D1 AX10 light microscope (Carl Zeiss Microscopy)

Procedures

  1. Bone explant culture

    1. Prepare washing and growth media before mouse sacrifice.

    2. Keep 10 ml washing medium on ice to keep the bones in until culture.

    3. Place the growth medium in a 37°C water bath.

    4. Sacrifice the mice by cervical dislocation (or any other appropriate method).

    5. Rinse the animal thoroughly with 10 ml 70% ethanol.

    6. Bone isolation (Time: 10 min/mouse, requires 2 scissors and 2 forceps)

      1. Incise the skin of the inguinal region using scissors and remove it entirely from the hindlimbs. Disconnect the hindlimbs (femur with attached tibia) from the trunk by cutting at the femoral head with scissors. Place the hindlimbs in a sterile 100-mm Petri dish (Note 3, see Figure 1A).

      2. Cut at the knee junction to separate the tibia from the femur. Remove the soft tissue using forceps and scissors. Avoid taking hair and fat (see Figure 1A).

      3. Place the bones (femurs and tibias) in a 50-ml conical tube containing 15 ml washing medium on ice until all the bones have been isolated (see Notes 4 and 5).

    7. Bone marrow cell removal (Time: 2 min/bone, requires 2 scissors and 2 forceps)

      1. Under a cell culture hood, place the bones into a dish containing 10 ml washing medium.

      2. Cut the epiphyses just below the end of the marrow cavity using sterile scissors.

      3. Place the diaphysis into an empty Petri dish.

      4. Insert a 25 G needle with a 1-ml syringe filled with 1 ml growth medium into the bone marrow cavity and flush out the bone marrow. Repeat this step at least 3 times, until the bone becomes white (Notes 6 and 7, see Figure 1A).

      5. Place the flushed bones into a 50-ml tube containing 5 ml ice-cold growth medium for periosteal cell isolation.

    8. Periosteal cell culture

      1. Remove the growth medium and wash the bones by rinsing 3 times with fresh growth medium.

      2. Place all 12 bones into a 60-mm TPP culture dish (in the center of the dish, each bone separated by 0.3 cm, see Figure 1B).

      3. Cover the bones by adding drops of growth medium at 37°C (around 1 ml in total, see Note 8).

      4. Place the bones in a humid CO2 incubator at 33°C or 37°C and 5% CO2 (see Note 9).

      5. Change the medium daily. Carefully aspirate the medium and replace with fresh growth medium. Place the dish back at 33°C or 37°C (see Notes 10 and 11). Cell migration usually starts after 2 or 3 days.

    9. When cell migration is observed (Figure 1C), add 2-3 ml growth medium. Change the medium daily.

    10. When PCs have sufficiently migrated from the explant (usually after 2 weeks), gently remove the explants from the dish using sterile forceps, wash once with 1× PBS, and add 5 ml fresh growth medium to cover the cells (Figure 1C).

    11. Allow the cells to grow for 4-5 more days. Change the medium daily.

    12. When PCs reach 80% confluence around the explant site, remove the medium and add 3 ml trypsin. Place the plate back into the incubator for 3 min. Detach PCs using a cell scraper and transfer the cells to a 50-ml Falcon tube containing 10 ml growth medium.

    13. Centrifuge at 300 × g for 10 min.

    14. Discard the supernatant and resuspend the cells in 10 ml growth medium. Plate the cells into a 100-mm TPP plate.

    15. Replace the medium every 2-3 days with fresh growth medium.

    16. When PCs reach 80% confluence, cells can be trypsinized for further passage (Figure 1D). For experiments, PCs can be used from P0 (Notes 12, 13, and 14).



      Figure 1. Steps of bone explant culture. (A) Steps of bone dissection. Left, hindlimb free of skin. Middle, tibia, and femur free of muscle. Right, tibia and femur after cutting the epiphyses and flushing the bone marrow. (B) Tibias and femurs plated in a culture dish. (C) After a few days, periosteal cells (PCs) migrate out of the bone explants into the dish. (D-E) PCs at P0 and P1.


  2. Flow cytometry analysis of PCs

    1. When PCs reach 80% confluence, remove medium and add 3 ml trypsin. Place the plate back into the incubator for 3 min. Detach PCs using a cell scraper, and transfer the cells into a 50-ml Falcon tube containing 10 ml growth medium.

    2. Centrifuge at 300 × g for 10 min.

    3. Resuspend cells in 10 ml growth medium.

    4. Filter through a 40-μm cell strainer.

    5. Count the living cells using Trypan Blue and process the number of cells needed for the experiment: 1.5 × 105 cells per tube.

    6. Centrifuge at 300 × g for 10 min.

    7. Resuspend the cells in 200 μl FACS medium per 1.5 × 105 cells.

    8. Split the cells into FACS tubes.

    9. Add antibody mix according to each tube and mix well (Notes 15 and 16).

    10. Incubate for 15 min on ice in the dark.

    11. Add 1 ml FACS medium to each tube.

    12. Centrifuge at 300 × g for 10 min.

    13. Remove the supernatant and resuspend the cells in 200 μl FACS medium.

    14. Add 0.5 μl Sytox Blue per tube immediately before analysis.

    15. Perform flow cytometry analysis using a BD LSR Fortessa (Figure 2).



      Figure 2. Flow cytometry analysis of periosteum-derived cells at P1. Blue curves represent FMO controls, and red curves represent experimental samples.


  3. In vitro differentiation

    1. In vitro adipogenesis

      1. Plate 1.5 × 105 PCs in a 6-well plate in duplicate.

      2. Allow the cells to reach 100% confluence in growth medium.

      3. When confluency is reached, induce adipogenesis by covering the cells with adipogenic medium.

      4. Incubate the cells at 37°C for up to 3 weeks; change adipogenesis medium twice a week.

      5. After 3 weeks of differentiation, proceed to Oil Red O staining for lipid droplets:

        Discard medium.

        Rinse gently twice with PBS.

        Fix cells with 3 ml 70% ethanol for 30 min at room temperature.

        Rinse gently twice with H2O.

        Cover the cells with 3 ml 60% isopropanol for 5 min.

        Discard isopropanol and stain with 3 ml filtered Oil Red O staining solution for 50 min at room temperature.

        Rinse twice with H2O.

        Counterstain nuclei with 3 ml hematoxylin for 1 min.

        Rinse twice with H2O.

        Leave H2O and take an image within 2 h using an inverted microscope (Figure 3).

    2. In vitro osteogenesis

      1. Plate 1.5 × 105 PCs in a 6-well plate in duplicate.

      2. Allow the cells to reach 100% confluence in growth medium.

      3. When confluency is reached, induce osteogenesis by covering the cells with osteogenic medium.

      4. Incubate the cells at 37°C for up to 3 weeks; change the osteogenic medium twice a week.

      5. After 3 weeks of differentiation, proceed to Alizarin Red staining for hydroxyapatite crystals.

        Discard medium.

        Rinse twice with PBS.

        Fix cells with 3 ml 70% ethanol for 30 min at room temperature.

        Rinse twice with H2O.

        Stain with 3 ml Alizarin Red S solution for 45 min at room temperature, under agitation and protected from light.

        Rinse twice with H2O.

        Allow the plate to dry before taking images (Figure 3).

    3. In vitro chondrogenesis

      1. Resuspend the cells at a concentration of 5 × 105 cells in 200 μl growth medium.

      2. Using a 200-μl pipet, seed the PCs in drops of 200 μl containing 5 × 105 cells. Place 3 drops in a 60-mm TPP culture dish.

      3. Carefully place the plates at 37°C (Note 17).

      4. Allow the cells to attach to the plate for 6-8 h.

      5. Check cell attachment before starting differentiation (leave longer if necessary).

      6. Remove growth medium and induce chondrogenesis by covering the cell drops with chondrogenic medium.

      7. Incubate the cells at 37°C for 3 days.

      8. Proceed to Alcian Blue staining for sulfated GAG:

        Discard medium.

        Rinse twice with PBS.

        Fix cells with 2% glutaraldehyde in H2O for 1 h at room temperature.

        Rinse with 0.1 M HCl.

        Stain with 1% Alcian Blue for 2 h at room temperature.

        Rinse twice with 0.1 M HCl.

        Allow the plate to dry before taking images (Figure 3).



      Figure 3. In vitro adipogenic, osteogenic, and chondrogenic differentiation of PCs before and after staining. Scale bars: before staining: 200 μm; staining: 50 μm (adipogenesis), 1 cm (osteogenesis), 2.5 mm (chondrogenesis).


  4. In vivo PC transplantation at the fracture site (Note 18)

    1. Tisseel matrix pellet formation

      1. When PCs reach 80% confluence, remove the medium and add 3 ml trypsin. Place the plate back into the incubator for 3 min. Detach PCs using a cell scraper and transfer the cells to a 50-ml Falcon tube containing 10 ml growth medium.

      2. Centrifuge at 300 × g for 10 min.

      3. Count the cells and keep only the number of cells needed for the experiment: 105 cells per host animal.

      4. Place 105 cells in a 1.5-ml sterile Eppendorf tube (one tube for each host animal). Centrifuge for 10 min at 300 × g and remove as much medium as possible.

      5. Prepare appropriate volumes of fibrin (F) and thrombin (T) diluted 1:4 in distilled water: 105 cells are resuspended in 15 μl F (diluted 1:4) + 15 μl T (diluted 1:4) per host animal (see Note 19).

      6. Add 15 μl diluted fibrin to the tubes containing cells. Resuspend by pipetting without forming bubbles.

      7. Add 15 μl diluted thrombin to form a solid gel (see Note 20).

      8. Allow the matrix to polymerize for 15 min on ice. If the pellet is well formed, it can be easily grabbed with the tip of forceps (Figure 4E).

      9. Keep the tubes at 4°C on ice until transplantation.

    2. Surgery for in vivo transplantation

      1. Anesthetize mice with intraperitoneal injection of 50 mg/kg ketamine and 1 mg/kg medetomidine (Note 21).

      2. Inject 0.1 mg/kg buprenorphine subcutaneously for analgesia.

      3. After 15 min, check the quality of anesthesia by foot pinching.

      4. Shave the right limb and sanitize using vetedine soap and solution or any other skin disinfectant solution (Figure 4A).

      5. Perform a 2-cm incision on the skin above the tibia using a sterile scalpel (Figure 4B).

      6. Expose the anterior tibial surface by carefully separating the muscle from the bone surface (Figure 4C).

      7. Create 3 holes at the mid-diaphysis perpendicular to the longitudinal axis of the tibia using a drill and a 0.4-mm drill bit.

      8. Induce an osteotomy by cutting the bone along the 3 holes with scissors (see Note 22) (Figure 4D).

      9. Gently position the Tisseel matrix pellet containing PCs at the fracture site between the two bone cortices (Figure 4F).

      10. Close the skin wound using 5-0 non-resorbable sutures.

      11. Revive the mice with an intraperitoneal injection of 1 mg/kg atipamezole.

      12. Place the mice on a 37°C heating pad until revived.

      13. Perform two additional subcutaneous injections of buprenorphine 0.1 mg/kg at 12 h and 24 h post-surgery.

    3. Histological analysis of the PC contribution to the callus

      1. Sacrifice the mice by cervical dislocation (or any other appropriate method) and rinse the limb with 70% ethanol (Note 23).

      2. Incise the skin and remove it entirely from the hindlimbs. Disconnect the femur and the tibia by cutting at the knee junction and at the feet. Remove the soft tissue around the callus using forceps and scissors (Note 24).

      3. Place the sample in a 15-ml Falcon tube containing 8 ml 4% ice-cold PFA for 24 h with constant shaking at 4°C.

      4. Remove the PFA solution, wash the sample with ice-cold PBS, and add 8 ml EDTA solution. Place at 4°C with constant shaking for 21 days; change the EDTA solution every other day (see Note 25).

      5. Remove the EDTA solution, wash the sample with ice-cold PBS, and add 8 ml cryoprotection solution. Place at 4°C for 24 h without shaking.

      6. Remove the cryoprotection solution and wash 3 times with ice-cold PBS.

      7. On dry ice, fill a plastic mold with Tissue Freezing Medium, embed the bone, and place the sample at -80°C.

      8. Cut the sample into 10 μm-thick sections using a cryostat.

      9. Stain the sections with Safranin O to allow cartilage visualization (Figure 4G):

        Allow the slides to dry for 30 min at room temperature.

        Hydrate the slides in PBS for 5 min.

        Place the slides in Weigert’s solution for 5 min.

        Wash in tap water for 3 min.

        Place the slides in Fast Green solution for 30 s.

        Place the slides in 1% acetic acid for 30 s.

        Place the slides in Safranin O solution for 30 min.

        Wash in distilled water for 3 min.

        Place the slides in 70% ethanol for 3 min.

        Place the slides in 95% ethanol for 3 min.

        Place the slides in 100% ethanol for 5 min, twice.

        Place the slides in Neo-Clear for 3 min, twice.

        Mount the slides with Neo-mount.

        Take images using a brightfield microscope.

      10. Mount the slides with DAPI to visualize the PC contribution to the callus (Figure 4H):

        Allow the slides to dry for 30 min at room temperature.

        Hydrate the slides in PBS for 5 min.

        Mount the slides with Fluoromount-G with DAPI.

        Take images using a fluorescence microscope.



      Figure 4. In vivo transplantation of PCs isolated from GFP-actin donors at the fracture site of wild-type hosts . After shaving and sanitizing the limb (A), an incision in the skin is performed (B) and the tibia is exposed (C, tibia is delineated by a dotted line) to induce a fracture (D). A Tisseel matrix pellet containing PCs (E) is transplanted at the fracture site (F, black arrow). (G) Representative image of a longitudinal callus section on day 14 post-fracture stained with SO. (H) GFP+ chondrocytes derived from PCs on an adjacent section of the callus. Scale bars: 1 mm (G) and 100 μm (H).

Notes

  1. We recommend using the hindlimbs (femur and tibia bones) of at least 3 mice per culture. Donor mice for PC culture should carry a reporter transgene in the C57BL/6J background in order to perform cell tracing following in vivo transplantation in C57BL/6J hosts.

  2. All procedures must be approved by the local Ethics Committee.

  3. Two sets of sterilized surgical tools (2 scissors and 2 forceps per set) are needed for bone explant culture: one for bone dissection and one for bone marrow removal under a culture hood. To avoid contamination, tools used to cut and remove the skin should not be used to touch bones and soft tissue. Bones should only be handled using forceps to avoid any contamination.

  4. Remove soft tissue gently along the bone using scissors, instead of pulling out tissues, to avoid detaching the periosteum from the cortex.

  5. Isolated bones can be kept on ice in washing medium for up to 2 h.

  6. Be careful not to drop the bones into the Petri dish containing the bone marrow cells in order to avoid contamination of PC cultures with BMSCs. Any bone dropped in the plate containing BMSCs should be discarded.

  7. BMSCs can be cultured from the flushed bone marrow. Centrifuge the medium containing the flushed bone marrow at 300 × g for 10 min, resuspend the pellet in growth medium, and plate in a 100-mm TPP culture dish. On subsequent days, wash the cells with PBS once a day to eliminate floating hematopoietic cells and obtain adherent bone marrow cells.

  8. When covering the bones with medium, drops covering each bone can touch and merge. However, be careful that the bones are not floating or detaching, as it is critical for bones to be in contact with the plate to allow for cell migration.

  9. The protocol was initially optimized using a 33°C incubator, but PC migration and growth are also observed when cultured in a 37°C incubator.

  10. Do not wash the bones and proceed gently to avoid moving bones that have started to attach to the bottom of the dish.

  11. Change the medium daily to avoid bone explant drying.

  12. We recommend using PCs between passages 0 and 2 for optimal cell growth.

  13. PCs can be frozen down for later use. For optimal cell viability after thawing, we recommend freezing cells in growth medium supplemented with 10% DMSO.

  14. As observed in Figure 2, primary cultures of PCs also contain hematopoietic cells. An additional step of cell sorting or antibody-based column depletion can be used to discard hematopoietic cells and increase the purity of periosteal stem/progenitor populations.

  15. Each antibody has to be titrated prior to the experiment.

  16. For each experiment, FMO (Fluorescence Minus One) controls can be included to determine positive vs. negative signals, and isotype controls can be used to check the specificity of the antibodies.

  17. After plating cell drops for chondrogenesis, handle the plates with caution, as the drop should remain intact to allow cell deposition at the right confluency to mimic a 3D environment.

  18. For tracing of PC contribution to the fracture callus, donor mice should express a reporter gene. As an example, we use GFP-actin mice in this protocol.

  19. Fibrin is a highly viscous solution; be careful to pipet the right volume of fibrin when diluting. If needed, cut the extremity of the tip for easier pipetting of the viscous solution.

  20. Coagulation is very fast. After adding thrombin, remove the tip from the gel within 2 s.

  21. Anesthetized mice should be kept on a 37°C heating pad to avoid temperature loss and improve well-being.

  22. Tibia can be cut without drilling holes, but drilling holes prevents bone fragments.

  23. The time of sacrifice should be decided according to the biological question. We recommend choosing days 7-14 post-fracture to observe the contribution to cartilage and days 14-21 to observe the contribution to bone.

  24. Be careful to remove enough soft tissue without affecting the callus structure, especially when collecting on days 3, 5, and 7 post-fracture.

  25. The pH of the EDTA solution must be between 7.4 and 7.6 to allow for proper decalcification.

Recipes

  1. Washing medium

    DMEM

    1% penicillin/streptomycin

  2. Growth medium

    α-MEM with GlutaMAX

    1% penicillin/streptomycin

    20% lot-selected fetal bovine serum

    10 ng/ml bFGF

  3. FACS medium

    PBS

    1% penicillin/streptomycin

    2% fetal bovine serum

  4. Adipogenic medium (make fresh)

    α-MEM with GlutaMAX

    1% penicillin/streptomycin

    10% fetal bovine serum

    0.1 μM dexamethasone

    100 μM indomethacin

    0.5 mM IBMX

    10 μg/ml insulin

  5. Oil Red O stock solution

    Stock solution: 300 mg Oil Red O powder in 100 ml 99% isopropanol

    Ready-to-use solution (make fresh):

    Mix 3 parts Oil Red O stock solution with 2 parts H2O

    Allow to sit at room temperature for 10 min

    Filter the staining solution through coffee filter paper

  6. Osteogenic medium (make fresh)

    α-MEM with GlutaMAX

    1% penicillin/streptomycin

    10% fetal bovine serum

    0.1 μM dexamethasone

    0.05 mM L-ascorbic acid

    10 mM β-glycerophosphate

  7. Alizarin Red S staining solution

    Alizarin Red S 0.2% in distilled water

    Adjust the pH to 4.1-4.3.

    The pH is critical; make fresh or check the pH if the solution is more than one month old.

    Store at 4°C.

  8. Chondrogenic medium (make fresh)

    DMEM high glucose

    1% penicillin/streptomycin

    10% fetal bovine serum

    0.1 μM dexamethasone

    100 μg/ml sodium pyruvate

    40 μg/ml L-proline

    50 μg/ml L-ascorbic acid

    50 mg/ml ITS

    10 ng/ml TGFβ1

  9. 1% Alcian Blue staining solution

    Prepare 5% Alcian Blue solution in PBS

    Dilute 5% Alcian Blue stock solution in 0.1 M HCl to obtain 1% Alcian Blue

  10. Cryoprotection solution

    30 g sucrose

    100 ml H2O

  11. Weigert’s solution (make fresh)

    100 ml solution A and 100 ml solution B

    Solution A:

    5.0 g hematoxylin

    500 ml ethanol 95%

    Solution B:

    20 ml 29% aqueous iron(III) chloride

    5 ml HCl

    475 ml distilled water

  12. Fast Green solution

    0.2 g Fast Green

    1 L distilled water

  13. Safranin O solution

    1 g Safranin O

    1 L distilled water

Acknowledgments

This work was supported by Fondation de l’Avenir, Osteosynthesis and Trauma Care Foundation, ANR-18-CE14-0033, NIAMS R01 AR072707 to C.C. A.J., S.P., and O.D.L were supported by Ph.D. fellowships from Paris University. This protocol was first described in Duchamp de Lageneste et al. (2018) and in Julien et al. (2020).

Competing interests

The authors declare no competing interests.

Ethics

All procedures involving animals were approved by the Creteil University (agreement #19295-2019052015468705) Ethical Committee.

References

  1. Arnsdorf, E. J., Luis M. J., Dennis, R. C. and Christopher, R. J. (2009). The periosteum as a cellular source for functional tissue engineering. Tissue Eng Part A 15(9): 2637-2642.
  2. Arthur, A. and Gronthos, S. (2020). Clinical Application of Bone Marrow Mesenchymal Stem/Stromal Cells to Repair Skeletal Tissue. Int J Mol Sci 21(24): 9759.
  3. Brownlow, H. C., Reed, A., Joyner, C., Simpson, A.H. (2000). Anatomical effects of periosteal elevation. J Orthop Res 18(3): 500-502.
  4. Chang, H. and Knothe Tate, M. L. (2012) Concise review: the periosteum: tapping into a reservoir of clinically useful progenitor cells. Stem Cells Transl Med 1(6): 480-491.
  5. Debnath, S., Yallowitz, A.R., McCormick, J., Lalani, S., Zhang, T., Xu, R., Li, N., Liu, Y. F., Yang, Y. S., Eiseman, M., Shim, J. H., Hameed, M., Healey, J. H., Bostrom, M. P., Landau, D. A., Greenblatt, M. B. (2018). Discovery of a periosteal stem cell mediating intramembranous bone formation. Nature 562(7725): 133-139.
  6. Duchamp de Lageneste, O., Julien, A., Abou-Khalil, R., Frangi, G., Carvalho, C., Cagnard, N., Cordier, C., Conway, S. J., Colnot, C. (2018). Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin. Nat Commun 9(1): 773.
  7. van Gastel, N., Torrekens, S., Roberts, S. J., Moermans, K., Schrooten, J., Carmeliet, P., Luttun, A., Luyten, F. P., Carmeliet, G. (2012). Engineering vascularized bone: osteogenic and proangiogenic potential of murine periosteal cells. Stem Cells 30(11): 2460-2471.
  8. Julien, A. Perrin, S., Duchamp de Lageneste, O., Carvalho, C., Bensidhoum, M., Legeai-Mallet, L., Colnot, C. (2020). FGFR3 in Periosteal Cells Drives Cartilage-to-Bone Transformation in Bone Repair. Stem Cell Reports 15(4): 955-967.
  9. Ortinau, L. C., Wang, H., Lei, K., Deveza, L., Jeong, Y., Hara, Y., Grafe, I., Rosenfeld, S. B., Lee, D., Lee, D., Scadden, D. T., Park, D. (2019). Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells. Cell Stem Cell 25(6): 784-796.e5.
  10. Wang, Q., Huang, C., Zeng, F., Xue, M., Zhang, X. (2010). Activation of the Hh pathway in periosteum-derived mesenchymal stem cells induces bone formation in vivo: implication for postnatal bone repair. Am J Pathol 177(6): 3100-3111.

简介

[摘要]覆盖骨骼外表面的骨膜含有骨骼干/祖细胞,可在骨骼修复过程中有效地形成软骨和骨骼。已经描述了几种基于刮骨和/或酶消化分离骨膜细胞的方法。在这里,我们描述了一种外植体培养的方法来分离骨膜-衍生的用于随后的干/祖细胞在体外和体内分析。使用该协议分离的骨膜细胞 (PC)表达间充质标记,可以在体外扩增,并在骨折部位进行体内移植后表现出很高的再生潜力,表明该协议可用于 PC 生产以用于新的细胞疗法。


[背景]骨再生是一个高效的过程。骨折后,骨骼干/祖细胞被激活并分化成软骨细胞和成骨细胞,形成软骨和骨骼以巩固骨折。骨骼肌干/祖细胞起源从所述骨本身包括骨髓和骨膜,以及从相邻的软组织。骨骼再生过程中骨骼干/祖细胞的不同来源表明这些细胞可以从各种来源获得用于干细胞治疗。由于它们的可及性,骨髓基质细胞 (BMSC) 是研究最多的(Arthur 和 Gronthos,2020);ħ H但是,它们的可变成骨能力亮点需要细胞的新来源能够contribut荷兰国际集团有效地在修补过程。最近的研究揭示了骨膜作为骨再生过程中干/祖细胞的重要来源的作用(Debnath等人,2018 年;Duchamp de Lageneste等人,2018 年;Ortinau等人,2019 年)。当移植到骨骨折部位,骨膜细胞(PC)的显示更高的再生潜能比BMSC小号和能纠正一个骨修复故障的能力(杜尚德Lageneste等人,2018;于连。等人,2020)。分离小鼠 PC 具有挑战性,因为骨膜是骨骼外表面上的一层非常薄的组织。之前已经描述了几种方法来分离小鼠的 PC,依赖于酶消化和骨膜刮除或剥离(Brownlow等人,2000 年;Arnsdorf等人,2009 年;Wang等人,2010 年;Chang 和 Knothe Tate,2012 年;van Gastel等人,2012 年)。在这里,我们描述了基于细胞MI分离的PC的方法从骨植格雷申而不消化或骨膜从分离的骨(图URE 1)。我们开发了这种方法来分析 PC 特性,以便与通常通过直接骨髓冲洗和电镀分离的 BMSC 进行直接比较。免费骨骼肌,骨骺的长骨,和骨髓被置于培养基,允许PC迁移和增殖。使用这种方案分离的PC表达间充质标记物(图URE 2),显示在体外脂肪形成,成骨,和软骨形成分化能力(图URE 3) ,和能够形成软骨和骨在体内移植在胫骨骨折的部位(图URE 4)。电脑日erefore维持其osteochondrogenic能力,为个人电脑的研究和提供新的潜在的观点基于细胞疗法IR使用。

关键字:骨膜, 骨再生, 骨骼干细胞/祖细胞, 体内细胞移植, 在体外分化

材料和试剂
 
猎鹰® 5 -毫升ř ound -b ottom编p olystyrene吨EST吨UBE小号(康宁,目录号:352235)
40 - μm 细胞过滤器(Fisher Scientific,目录号:352340)
锥形管,15 - ml和50 -毫升中(Falcon,目录号小号:352097 [15毫升]和352070 [50毫升]或等同物)
25 G针(Terumo,目录号:AN * 2516R1)
1 - ml 注射器(Terumo,目录号:SS+01H1)
60-mm TPP 培养皿(TPP,目录号:93060)
100-mm TPP 培养皿(TPP,目录号:93100)
6 -井板(TPP,目录号:009206)
Greiner Bio-One 培养皿(细菌培养皿;Dutscher,目录号:633185)
10次-ml和25 -毫升的移液管(Dutscher,目录号小号:357551 [10毫升]和357535 [25毫升]或等同物)
细胞刮刀(TPP,目录号:99010 或等效物)
Kova ®载玻片(Fisher Scientific,目录号:22-270141)
猎鹰® 5 -毫升ř ound -b ottom编p olystyrene吨EST吨UBE小号(康宁,目录号:352235)
玻璃小号大环内酯类,正电荷防(热费舍尔,目录号:J1800AMNZ)
盖玻片(Labellians,目录号:LCO2460M)
对于骨膜来源的细胞培养物:4至8 -周-老的小鼠在C57BL / 6J背景(见注1)
主机用于体内细胞移植:10〜14 -周-老的小鼠在C57BL / 6J背景(见注2)
DMEM(Life Technologies,目录号:11966-025)
青霉素小号treptomycin(P / S; Life Technologies公司,目录号:15140-122)
α-改良Eagle的中号edium(α-MEM含GlutaMAX; Life Technologies公司,目录号:32561-029)
很多选择˚F等人b绵羊小号erum(FBS; Life Technologies公司,目录号:10270-106)
重组小鼠基本˚F ibroblast克rowth ˚F演员(bFGF的; R d,目录号:3139FB / CF)
台盼乙略染色(Life Technologies公司,目录号:15250-061)
胰蛋白酶-EDTA(0 . 25%),酚红(Life Technologies,目录号:25200056)
地塞米松(Sigma-Aldrich,目录号:D8893)
人类我nsulin溶液(Sigma-Aldrich公司,目录号:I9278)
吲哚美辛(Sigma-Aldrich,目录号:I7378)
3-异丁基-1-米ethylxantine(IBMX,Sigma-Aldrich公司,目录号:I5879)
油- [R编O(Sigma-Aldrich公司,目录号:O0625)
异丙醇(Sigma-Aldrich,目录号:I9516)
L-抗坏血酸(Sigma-Aldrich,目录号:A8960)
β-甘油磷酸酯(Sigma-Aldrich,目录号:G9422)
茜素R ed S(Sigma-Aldrich,目录号:A5533)
DMEM 高葡萄糖(Life Technologies,目录号:31966-021)
丙酮酸钠(Sigma-Aldrich,目录号:P5280)
L-脯氨酸(Sigma-Aldrich,目录号:P5607)
胰岛素吨ransferrin-小号裂果硒(ITS ; Sigma-Aldrich公司,目录号:I1884)
TGF - β1 (Sigma-Aldrich,目录号:T7039)
阿尔辛蓝(Sigma-Aldrich,目录号:A5268)
戊二醛(默克,目录号:1-04239-0250)
盐酸(Sigma-Aldrich,目录号:H1758)
磷酸盐缓冲盐水(PBS;Life Technologies,目录号:14190-094)
乙醇(绝对乙醇≥99.8% ;VWR,目录号:20821.365)
苏木精(Sigma-Aldrich,苏木精溶液,Harris Modified,目录号:HHS32-1L)
Sytox Blue 死细胞染色剂(Life Technologies,目录号:S34857)
Tisseel m at rix(凝血酶和纤维蛋白溶液,Baxter,目录号:3400894252443)
S无菌蒸馏水(Life Technologies,目录号:15230162)
Bupr ë norphine(Centravet,目录号:BUP001)
Atipame zole (Centravet,目录号:ANT201)
氯胺酮(Centravet,目录号:KET205)
美托咪定(Centravet,目录号:DOM003)
Vetedine Savon(Vetoquinol,目录号:2608436 7/1992)
Vetedine小号olution(Vetoquinol,目录号:45768891992分之5)
多聚甲醛 4%(PFA ; Clinisciences,目录号:sc-28169 2)
蔗糖(VWR,目录号:443815S)
EDTA溶液0.5 M(Euromedex,目录号:EU0084)
组织冷冻培养基(MMFrance,目录号:F/TFM-C)
Neo-Clear(Sigma-Aldrich,目录号:109843)
无水苏木精(Sigma-Aldrich,目录号:109843)
铁(III)Ç hloride,97%(Sigma-Aldrich公司,目录号:157740)
Fast Green(Sigma-Aldrich,目录号:F7252)
Safranin O(Sigma-Aldrich,目录号:S2255)
Neo-mount(Sigma-Aldr ich,目录号:109016)
带有 DAPI 的 Fluoromount-G 安装介质(Thermo Fisher,目录号:00-4959-52)
PE-Cy TM 7大鼠抗小鼠CD31抗体(稀释1/400 ;BD Bioscience,目录号:561410 )
PE-Cy TM 7大鼠抗小鼠CD45抗体(稀释1/400 ;BD Bioscience,目录号:552848 )
PE-Cy TM 7 大鼠抗小鼠 CD11b 抗体(稀释 1/400 ;BD Bioscience,目录号:552850 )
BV650大鼠抗小鼠Ly-6A/E抗体(稀释1/200 ;BD Bioscience,目录号:740450 )
PE仓鼠抗小鼠CD29抗体(稀释1/200 ;Miltenyi,目录号:130-102-994 )
 
设备
 
手术钳 (× 4)(Dumont AA 钳;FST,目录编号:11210-20,或同等产品)
手术剪(× 4)(细剪刀ToughCut ® 11毫米; FST,猫考勤号:14058-11,或等同物)
BD LSRFortessa机(Becton Dickinson )
细胞培养无菌罩
带温度控制的离心机
带温度控制的水浴
CO 2培养箱设置为 5% CO 2和33°C 或 37°C
摇床(VWR,型号:迷你章动式,3D 混合器)
Drill (Dremel, cat alog number: 8050-15)
钻头(0.4 毫米)
W/C3 NDL Silk BL 编织缝线(Havard Apparatus,猫编号:72-3318)
暖气p广告(哈佛仪器,猫考勤号:55-7033)
鼠标割草机(Kerbl,cat alog 编号:GT416)
无菌手术刀(Dutscher,猫编号:132622)
低温恒温器(Leica Biosystems)
MM35P刀片(MMFrance,F/MM35P)
Zeiss Imager D1 AX10 光学显微镜(Carl Zeiss Microscopy)
 
程序
 
骨外植体培养
准备清洗和增长MEDI一个鼠标处死前。
保持冰10毫升清洗介质,以保持在骨骼中,直到文化。
将生长培养基置于 37°C 水浴中。
牺牲了通过颈椎脱位(或任何其它适当的方法)的小鼠。
用 10 毫升 70% 乙醇彻底冲洗动物。
骨隔离(时间:10 分钟/鼠标,需要 2 把剪刀和 2 个镊子)
用剪刀切开腹股沟区域的皮肤,并将其从后肢上完全切除。用剪刀剪断股骨头,将后肢(附有胫骨的股骨)与躯干断开。放置在一个后肢无菌100毫米P ETRI培养皿(注3,参见图URE 1A)。
切口在膝盖结分离的胫骨从该股骨。使用镊子和剪刀去除软组织。避免摄取头发和脂肪(见图1A)。
放置骨(股骨小号和胫骨小号在50)-含有15毫升锥形管米升冰上洗涤介质,直到所有的骨已经被分离(见注小号4和5)。
骨髓细胞去除(时间:2 分钟/骨,需要 2 把剪刀和 2 个镊子)
下细胞培养罩,将骨头到含有10μm的培养皿升洗涤介质。
使用无菌剪刀在骨髓腔末端下方切开骨骺。
将 diap hysis 放入空的培养皿中。
插入25号针用一个1 -毫升注射器装入1ml生长培养基到骨的骨髓腔和冲洗出的骨的骨髓。重复此步骤至少 3 次,直到骨头变白(注释 6 和 7,见图1A )。
放置冲洗骨头成50 -包含骨膜细胞分离5毫升冰冷生长培养基毫升管。
骨膜细胞培养
移除所述生长培养基和洗涤的漂洗骨头3用新鲜的生长培养基中次。
将所有 12 根骨头放入 60 毫米 TPP 培养皿中(在培养皿的中心,每根骨头相隔 0.3 厘米,见图1B)。
覆盖的通过在37℃下加入生长培养基的滴骨(约1毫升总,见注8)。
放置的骨头在潮湿CO 2培养箱中在33 ℃下或37 ℃下和5%CO 2 (见注9)。
改变的日常中。小心地吸出培养基并更换为新鲜的生长培养基。将培养皿放回 33°C 或 37 °C (见注释 10 和 11)。细胞迁移通常在2或3天后开始。
当细胞迁移观察(图URE 1C ),加入2-3毫升生长培养基中。每天更换培养基。
当电脑已经充分地从外植体(通常是后2周)迁移,使用无菌镊子轻轻取出从盘的外植体,用1洗一次× PBS ,并加入5米升新鲜生长培养基以覆盖细胞(图URE 1C) .
让细胞再生长 4-5 天。改变的日常中。
当个人电脑达到80%confluenc Ë围绕植位点,除去该介质,并加入3 ml胰蛋白酶。将板放回培养箱中 3 分钟。使用分离的PC细胞刮刀,将细胞转移到50 -毫升˚F爱尔康TU可以含有10毫升生长培养基中。
以 300 × g离心10 分钟。
弃去上清液,将细胞重悬在 10 ml 生长培养基中。将细胞板放入 100 - mm TPP 板中。
每 2-3 天用新鲜的生长培养基更换培养基。
当个人电脑达到80%confluenc ë ,可以将细胞用胰蛋白酶处理以进一步通道(图URE 1D)。对于实验小号,个人电脑可以从可使用P 0(注12,13 ,和14)。
 
 
图1 。骨外植体培养步骤。(A) 骨解剖步骤。左,后肢没有皮肤。中东,牛逼IBIA ,和股骨免费的肌肉。对了,牛逼IBIA和股骨切割骨骺和冲洗骨髓后。(B) 胫骨和股骨镀在培养皿中。(C) 几天后,骨膜细胞 (PC) 从骨外植体迁移到培养皿中。(DE) P 0 和P 1处的 PC 。
 
PC 的流式细胞术分析
当个人电脑达到80%confluenc ë ,除去培养基并加入3 ml胰蛋白酶。将板放回培养箱中 3 分钟。使用分离的PC一个细胞刮刀,并将细胞转移到50 -毫升˚F爱尔康TU可以含有10毫升生长培养基中。
以300 × g离心10 分钟。
在 10 ml 生长培养基中重悬细胞。
过滤器通过一个40 - μ米细胞滤网。
算的使用台盼活细胞乙略和处理实验所需的细胞数:1.5 × 10 5每管细胞。
以300 × g离心10 分钟。
重悬的在200个细胞μ升每1.5 FACS培养基× 10 5细胞。
分裂细胞小号进入FACS管。
根据每管添加抗体混合物并混合均匀(注释 15 和 16 )。
Incub吃了在黑暗中冰15分钟。
向每管中加入 1 ml FACS 培养基。
以300 × g离心10 分钟。
去除上清并将细胞重新悬浮在200 μ升FACS平台。
添加0.5 μ升每管SYTOX蓝立即分析之前。
执行使用BD LSR Fortessa(图流式细胞分析URE 2)。
 
 
图2 。在P 1处对骨膜衍生细胞进行流式细胞术分析。蓝色曲线代表 FMO 对照,红色曲线代表实验样品。
 
体外分化
体外脂肪生成
将 1.5 × 10 5 PC置于 6孔板中,一式两份。
允许细胞以达到100%汇合于生长培养基中。
当达到汇合时,通过用脂肪生成培养基覆盖细胞来诱导脂肪生成。
孵育所述细胞在37℃下进行长达3周; 每周更换两次脂肪生成培养基。
分化 3 周后,继续对脂滴进行油R ed O 染色:
丢弃介质。
用 PBS 轻轻冲洗两次。
在室温下用 3 m l 70% 乙醇固定细胞30 分钟。
用 H 2 O轻轻冲洗两次。
覆盖3细胞米升60%异丙醇5分钟。
丢弃异丙醇和染色用3米升过滤油- [R编O代表在室温下50分钟染色溶液。
用 H 2 O冲洗两次。
复染剂与3个核米升苏木1分钟。
用 H 2 O冲洗两次。
离开ħ 2 O和拍摄N个图像在2小时内使用倒置显微镜(图URE 3)。
体外成骨
将 1.5 × 10 5 PC 置于 6孔板中,一式两份。
允许细胞以达到100%汇合于生长培养基中。
当达到汇合时,通过用成骨培养基覆盖细胞来诱导成骨。
孵育所述细胞在37℃下进行长达3周; 改变了成骨细胞培养基每周两次。
3周分化后,前往茜素- [R ED染色为羟基磷灰石晶体。
丢弃介质。
用 PBS 冲洗两次。
在室温下用 3 m l 70% 乙醇固定细胞30 分钟。
用 H 2 O冲洗两次。
染色用3米升茜素ř编氏溶液,在室温下45分钟,在搅拌下并避光。
用 H 2 O冲洗两次。
在拍摄图像之前让印版干燥(图3)。
体外软骨形成
重悬的细胞以一个5个的浓度× 10 5细胞在200 μ升生长培养基中。
使用200 - μ升在200滴吸管,种子中的个人电脑μ升含有5 × 10 5细胞。在 60 毫米 TPP 培养皿中放置 3 滴。
小心地将板置于 37°C(注 17)。
允许细胞附着到板6 - 8小时。
在开始分化之前检查细胞附着(如有必要,留更长的时间)。
去除生长培养基并通过用软骨形成培养基覆盖细胞滴来诱导软骨形成。
将细胞在 37°C 下孵育3 天。
前往阿尔乙略染色的硫酸化GAG:
丢弃介质。
用 PBS 冲洗两次。
在室温下用 H 2 O 中的2% 戊二醛固定细胞1 小时。
用 0.1 M HCl 冲洗。
在室温下用 1% Alcian B lue染色2 小时。
用 0.1 M HCl 冲洗两次。
在拍摄图像之前让印版干燥(图3)。
 
 
图3 。体外脂肪,骨,和PC的软骨细胞分化前和染色后。比例尺:染色前:200 μ m ;染色:50 μ m(脂肪生成),1 cm(成骨),2 . 5毫米(软骨形成)。
 
骨折部位的体内PC移植(注18)
Tisseel 基质颗粒形成
当个人电脑达到80%confluenc Ë ,除去所述培养基并加入3 ml胰蛋白酶。将板放回培养箱中 3 分钟。使用分离的PC一个细胞刮刀,将细胞转移到50 -毫升˚F爱尔康TU可以含有10毫升生长培养基中。
以300 × g离心10 分钟。
计数的细胞,只保留所需要的实验的细胞数量:10个5每宿主动物细胞。
放置10个5细胞在一个1 。5 - ml 无菌E ppendorf 管(每只宿主动物一个管)。以300 × g离心 10 分钟,并尽可能去除培养基。
准备的适当体积˚F ibrin(F)和吨hrombin(T)稀释4:1在蒸馏水中:10个5重悬细胞在15 μ升F(稀释1:4 )+ 15 μ升T(稀释1:4 )每个宿主动物(见注释 19)。
添加15 μ升稀释˚F ibrin到的含有细胞的管。通过移液重悬而不形成气泡。
添加15 μ升稀释吨hrombin以形成固体凝胶(见注20 )。
允许所述基质以聚合在冰上15分钟。如果沉淀阱形成的,它可以被容易地与镊子的顶端抓起小号(图URE 4E)。
保持所述管在4°C在冰上直到移植。
手术体内移植
腹腔注射 50 mg/kg k etamine 和 1 mg/kg m edetomidine (注 21)麻醉小鼠。
皮下注射0.1 mg/kg b丙诺啡镇痛。
15 分钟后,通过捏脚检查麻醉质量。
剃右肢和使用vetedine肥皂和溶液或任何其它皮肤消毒液消毒(图URE 4A )。             
执行2 -上使用无菌解剖刀胫骨上方的皮肤厘米的切口(图URE 4B) 。
通过仔细地分离骨表面肌肉暴露胫骨前表面(图URE 4C) 。
创建垂直于使用钻头和0胫骨的纵向轴线在中间骨干3个孔4 -米米钻头。
通过切割用剪刀沿3个孔的骨(见注22)诱导的截骨术(图URE 4D)。
轻轻地定位TISSEEL米ATRIX粒料containin克电脑在骨折部位的两个骨皮层之间(图URE 4F )。
关闭皮肤用5-0无创伤-可吸收缝线。
用 1 毫克/公斤的替美唑腹腔注射复活小鼠。
将小鼠放在 37°C 的加热垫上,直到苏醒。
执行两个额外的皮下注射b在12 uprenorphine 0.1毫克/千克ħ和24小时后的手术。
PC对愈伤组织贡献的组织学分析
通过颈椎脱位(或任何其他适当的方法)处死小鼠,并用 70% 乙醇冲洗肢体(注 23)。
切开皮肤并将其完全从后肢上取下。断开股骨和通过在膝盖结切割和胫骨在脚。使用镊子和剪刀去除愈伤组织周围的软组织(注 24)。
将样品在一个15 -装有8毫升4%冰冷的PFA与恒定在4℃振荡24小时毫升Falcon管中。
除去PFA溶液,洗净用冰冷的样品-冷的PBS ,并且,加入8毫升EDTA溶液。置于 4°C 并不断摇动 21 天;每隔一天更换一次 EDTA 溶液(见注释 25)。
除去EDTA溶液,洗净用冰冷的样品-冷的PBS ,并且,加入8毫升冷冻保护溶液。在 4°C 下放置 24 小时,不要摇晃。
去除冷冻保护溶液并用冰冷的 PBS 洗涤 3 次。
在干冰上,用组织冷冻培养基填充塑料模具,嵌入骨头,并将样品置于 -80°C。
切割样品,以10 μ米-使用低温恒温器厚的切片。
染色的番红区段O操作允许软骨可视化(图URE 4G) :
允许载片干燥为在室温下30分钟。
将 PBS 中的幻灯片水合5 分钟。              
将幻灯片放在 Weigert 的解决方案中 5 分钟。
用自来水冲洗 3 分钟。
将幻灯片放入快速绿色溶液中 30 秒。
置于1%乙酸FO的幻灯片- [R 30秒。
将载玻片放入番红 O 溶液中 30 分钟。
用蒸馏水洗涤 3 分钟。
将幻灯片放入 70% 乙醇中 3 分钟。
将幻灯片放入 95% 乙醇中 3 分钟。
将载玻片放入 100% 乙醇中 5 分钟,两次。
将载玻片置于 Neo-Clear 中 3 分钟,两次。
使用 Neo-mount 安装幻灯片。
使用明场显微镜拍摄图像。
安装的幻灯片用DAPI可视化的愈伤组织的PC贡献(图URE 4H) :
允许载片干燥为在室温下30分钟。
将 PBS 中的幻灯片水合5 分钟。             
使用带有 DAPI 的 Fluoromount-G 安装幻灯片。
使用荧光显微镜拍摄图像。
 
 
图4 。体内分离自个人电脑的移植GFP肌动蛋白在野生的骨折部位供体-型的主机。在对肢体 (A) 进行剃须和消毒后,在皮肤上进行切口(B) 并暴露胫骨 (C,胫骨被虚线整齐) 以诱导骨折 (D)。将含有 PC (E) 的 Tisseel 基质颗粒移植到骨折部位 (F, 黑色箭头)。(G)的代表图像一个纵向愈伤组织部分上14天后骨折SO染色。(H) 来自愈伤组织相邻部分上的PC 的 GFP+ 软骨细胞。比例尺s :1 毫米(G)和 100微米(H)。
 
笔记
 
我们建议使用每个培养至少3 只小鼠的后肢(股骨和胫骨)。用于 PC 培养的供体小鼠应在 C57BL/6J 背景中携带报告基因转基因,以便在 C57BL/6J 宿主体内移植后进行细胞追踪。
所有的程序都必须由当地批准Ë THIC小号Ç ommittee。
两组都需要骨移植培养无菌外科手术工具(2个剪刀和每套2个钳子):一个用于骨解剖,一个用于下骨髓去除一个培养罩。为避免污染,不应使用用于切割和去除皮肤的工具来接触骨骼和软组织。只能使用镊子处理骨头以避免任何污染。
用剪刀沿骨轻轻取出软组织,而不是拉出组织,以避免分离的从皮层骨膜。
分离的骨头可以在洗涤介质中的冰上保存长达 2 小时。
要小心,不要骨头掉入P含有骨髓细胞培养皿ETRI为了给PC文化避免污染小号与骨髓基质干细胞。任何掉在含有 BMSCs 的板上的骨头都应该被丢弃。
BMSCs 可以从冲洗的骨髓中培养。将含有冲洗骨髓的培养基以300 × g离心10 分钟,将沉淀重悬在生长培养基中,并在 100 毫米 TPP 培养皿中培养。在随后的日子里,每天用 PBS 清洗一次细胞,以消除漂浮的造血细胞并获得粘附的骨髓细胞。
当覆盖的中等骨头,滴覆盖每个骨骼可以触摸和合并。然而,要注意的是在骨头不浮动或拆卸,因为它是至关重要的骨头以与所述板接触,以允许对细胞迁移。
该协议使用33℃培养箱中最初优化,但PC迁移和生长是在37℃的培养箱中培养时也观察到了。
不要洗骨头,轻轻地进行,以避免移动该骨头已经开始编到连接到该盘的底部。
每天更换培养基以避免骨外植体干燥。
我们建议在第 0 代和第 2 代之间使用 PC以获得最佳细胞生长。
PC 可以冻结以备后用。为了在解冻后获得最佳细胞活力,我们建议在补充有 10% DMSO 的生长培养基中冷冻细胞。
如图2中观察到的,原代培养š电脑还包含造血细胞。甲n中的细胞分选或基于抗体的柱耗尽的dditional步骤可以用于丢弃造血细胞和增加的骨膜干细胞/祖细胞群的纯度。
必须在实验前滴定每种抗体。
对于每个实验,可以包括 FMO(荧光减一)对照来确定阳性与. 负信号S,和同种型对照可用于检查的抗体的特异性。
在电镀细胞滴进行软骨形成后,小心处理板,因为滴应保持完整,以允许细胞沉积在正确的汇合处以模拟 3D 环境。
为了追踪 PC 对骨折愈伤组织的贡献,供体小鼠应表达报告基因。例如,我们在本协议中使用 GFP-肌动蛋白小鼠。
纤维蛋白是一种高粘性溶液;小心吸管的右音量˚F稀释时ibrin。如果需要,切掉尖端的末端,以便更容易地吸取粘性溶液。
凝固速度非常快。加入后吨hrombin ,除去2秒内从凝胶尖端。
麻醉小鼠应保持在 37°C 的加热垫上,以避免温度损失并改善健康状况。
无需钻孔即可切割胫骨,但钻孔可防止碎骨。
牺牲时间应根据生物学问题来决定。我们建议选择骨折后第7 - 14天观察对软骨的贡献,选择第 14 - 21天观察对骨骼的贡献。
小心而不影响愈伤组织结构,以除去足够的软组织,特别是收集当上天小号3,5 ,和7后-断裂。
EDTA 溶液的 pH 值必须介于 7.4和 7.6之间,以便进行适当的脱钙。
 
食谱
 
洗涤介质
DMEM
1%p enicillin /小号treptomycin
生长培养基
α -带有GlutaMAX 的MEM
1%p enicillin /小号treptomycin
20%很多-选择˚F等人b绵羊小号erum
10 纳克/毫升 bFGF
流式细胞仪培养基
PBS
1%p enicillin /小号treptomycin
2%˚F等人b绵羊小号erum
成脂培养基(新鲜)
α -带有 GlutaMAX 的 MEM
1%p enicillin /小号treptomycin
10%˚F等人b绵羊小号erum
0.1 μ M d依他米松
100 μ中号我ndomethacin
0.5 毫米 IBMX
10 μ微克/毫升我nsulin
石油[R编Ø原液
原液:300 毫克油R ed O 粉末在 100 毫升 99% 异丙醇中
准备-到-使用的解决方案(做新鲜):
将 3 份 Oil R ed O 原液与 2 份 H 2 O 混合
允许在室温下静置 10 分钟
过滤染色液通过咖啡过滤纸
成骨培养基(新鲜)
带有 GlutaMAX 的 α-MEM
1%p enicillin /小号treptomycin
10%˚F等人b绵羊小号erum
0.1 μ M d依他米松
0.05 mM L-抗坏血酸
10mM的β -甘油
茜素ř编S染色溶液
茜素红 S 0.2% 蒸馏水
调节pH至4.1 - 4.3。
pH 值很关键;让新鲜或检查的pH值,如果SOLUT离子超过一个月大。
储存在 4°C。
软骨形成培养基(新鲜)
DMEM高糖
1%p enicillin /小号treptomycin
10%˚F等人b绵羊小号erum
0.1 μ M d依他米松
100 μ微克/毫升小号裂果丙酮酸
40 μ g/ml L-脯氨酸
50 μ g/ml L-抗坏血酸
50 毫克/毫升 ITS
10 纳克/毫升 TGFβ1
1% Alcian B lue 染色液
制备5%爱茜乙在PBS略溶液
在 0.1 M HCl 中稀释 5% Alcian B lue 库存溶液以获得 1% Alcian B lue
防冻液
30 克蔗糖
100 毫升 H 2 O
魏格特的解决方案(新鲜)
100 毫升溶液 A 和 100 毫升溶液 B
解决方案一:
              5.0克ħ ematoxylin
              500毫升Ë THANOL 95%
解决方案B :
              20毫升29%含水我RON(III)Ç hloride
              5毫升盐酸
              475毫升蒸馏瓦特亚特
快速绿色解决方案
0.2克快速ģ颖
1升蒸馏水
番红O溶液
1 克番红 O
1升蒸馏水
 
致谢
 
通过基金会DE L'艾文莉,接骨和创伤护理基金会,ANR-18-CE14-0033,NIAMS R01 AR072707到CCAJ,SP这项工作是支持的,ODL由博士和支持。d 。巴黎大学的奖学金。该协议首先在 Duchamp de Lageneste等人中描述。( 2018 )和 Julien等人。(2020 年)。
 
利益争夺
 
a uthors 声明没有竞争利益s 。
 
伦理
 
所有涉及动物的程序都得到了克雷泰尔大学(协议 #19295-2019052015468705)伦理委员会的批准。
 
参考
 
Arnsdorf, EJ, Luis MJ, Dennis, RC 和 Christopher, RJ (2009)。骨膜作为功能性组织工程的细胞来源。组织工程 A 部分15(9) :2637 - 2642。
Arthur, A. 和 Gronthos, S. (2020) 。骨髓间充质干/基质细胞修复骨骼组织的临床应用。Int J Mol Sci 21(24) :9759 。
Brownlow, HC, Reed, A., Joyner, C., Simpson, AH (2000)。骨膜抬高的解剖学效应。J Orthop Res 18(3) :500 - 502。
Chang, H. 和 Knothe Tate, ML (2012)简明评论:骨膜:利用临床有用的祖细胞库。干细胞翻译医学1(6) :480 - 491。
Debnath, S., Yallowitz, AR, McCormick, J., Lalani, S., Zhang, T., Xu, R., Li, N., Liu, YF, Yang, YS, Eiseman, M., Shim, JH , Hameed, M., Healey, JH, Bostrom, MP, Landau, DA, Greenblatt, MB (2018)。发现介导膜内骨形成的骨膜干细胞。自然562(7725) :133 - 139。
Duchamp de Lageneste, O., Julien, A., Abou-Khalil, R., Frangi, G., Carvalho, C., Cagnard, N., Cordier, C., Conway, SJ, Colnot, C. (2018) . 骨膜含有由骨膜素控制的具有高骨再生潜力的骨骼干细胞。国家通讯社9(1):773。
van Gastel, N., Torrekens, S., Roberts, SJ, Moermans, K., Schrooten, J., Carmeliet, P., Luttun, A., Luyten, FP, Carmeliet, G. (2012)。工程血管化骨:小鼠骨膜细胞的成骨和促血管生成潜力。干细胞30(11) :2460 - 2471。
Julien, A. Perrin, S., Duchamp de Lageneste, O., Carvalho, C., Bensidhoum, M., Legeai-Mallet, L., Colnot, C. (2020)。骨膜细胞中的 FGFR3 在骨修复中驱动软骨到骨的转化。干细胞报告15(4) :955 - 967。
Ortinau, LC, Wang, H., Lei, K., Deveza, L., Jeong, Y., Hara, Y., Grafe, I., Rosenfeld, SB, Lee, D., Lee, D., Scadden, DT, Park, D. (2019)。功能不同的 Mx1+αSMA+ 骨膜骨骼干细胞的鉴定。细胞 干细胞25(6) :784-796.e5。
Wang, Q., Huang, C., Zeng, F., Xue, M., Zhang, X. (2010)。骨膜来源间充质干细胞中 Hh 通路的激活诱导体内骨形成:对出生后骨修复的影响。Am J Pathol 177(6) : 3100 - 3111。
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引用:Perrin, S., Julien, A., Duchamp de Lageneste, O., Abou-Khalil, R. and Colnot, C. (2021). Mouse Periosteal Cell Culture, in vitro Differentiation, and in vivo Transplantation in Tibial Fractures. Bio-protocol 11(15): e4107. DOI: 10.21769/BioProtoc.4107.
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