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May 2019

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Testing Bone Formation Induction by Calvarial Injection Assay in vivo
体内颅骨注射分析检测骨形成   

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Abstract

Bone formation occurs during embryogenesis, skeletal growth and during the process of skeletal renewal throughout life. In the process of bone formation, osteoblasts lay down a collagen-containing matrix, termed osteoid, which is gradually hardened by incorporation of mineral crystals. Although osteoblasts can be induced to differentiate and to deposit mineral in culture, this system does not always provide results that reflect the ability of agents to stimulate bone formation in vivo. This protocol describes a rapid and reliable method for testing local administration of agents on bone formation in vivo. In this method, mice are injected with the agent of question for 5 successive days. Fluorochrome labels are injected prior to, and after agents used for testing, and samples are collected and analysed by undecalcified bone histology and histomorphometry. This provides a robust method for assessing the ability of agents to stimulate bone formation, and if a short-term modification is used, can also be used for testing gene responses in bone to the same stimuli.

Keywords: Osteoblast (成骨细胞), Osteocyte (骨细胞), Bone formation (骨形成), Cytokines (细胞因子), Growth factors (生长因子)

Background

Bone formation is a fundamental process required for the development of the skeleton, for the progression of skeletal growth and to maintain bone structure throughout life as the skeleton is constantly remodeled. Bone formation is a process that occurs by three steps: (1) deposition of collagen-rich osteoid, (2) mineralization of that osteoid which occurs by deposition of bioapatite crystals around the collagen fibres, and (3) maturation of the mineralized bone substance (Blank and Sims, 2019). These three steps are mediated by two cell types derived from a common progenitor. Osteoblasts deposit osteoid and initiate mineralization, and as the osteoid is deposited, some osteoblasts become embedded within the bone matrix and differentiate into osteocytes (Dallas and Bonewald, 2010); these embedded cells also control the extent and nature of mineralization of bone (Vrahnas et al., 2019).

Identifying agents that can stimulate bone formation is critical for understanding the biology of the skeleton, and for developing new agents that can be used to promote bone formation in conditions of bone fragility such as osteoporosis, osteogenesis imperfecta, and osteomalacia, and to develop agents that can promote fracture healing or to stimulate bone formation in surgical interventions. Typically, the simplest system to test such agents is to use primary cultured osteoblasts (Orriss et al., 2014), or to differentiate stromal cell lines such as MC-3T3-E1 (Quarles et al., 1992) and Kusa 4b10 cells (Allan et al., 2003) in osteoblastogenic media. These systems are commonly used to study osteoblast differentiation by assessing effects of various agents on mRNA levels of osteoblast marker genes, alkaline phosphatase enzyme activity, and mineral deposition (Allan et al., 2003; McGregor et al., 2010; Walker et al., 2010). However, these systems do not always reflect the in vivo response. A clear example is the contrast between early in vivo works showing that leukemia inhibitory factor stimulates bone formation (Metcalf and Gearing, 1989; Cornish et al., 1993), and in vitro tests in which leukemia inhibitory factor could both stimulate and inhibit osteoblast differentiation (Malaval et al., 1995; Malaval and Aubin, 2001; Malaval et al., 2005; Falconi and Aubin, 2007).

In this protocol, we describe the use of a calvarial injection model which tests the ability of agents to stimulate bone formation in vivo. This method requires only small amounts of the stimulatory agent, and was first described by Cornish (Cornish et al., 1993) to resolve the controversy of whether leukemia inhibitor factor could promote bone formation. We have adapted the Cornish method to include calcein labelling to allow measurement of bone formation, and we have made use of it to test a range of cytokines including oncostatin M (Walker et al., 2010), cardiotrophin-1 (Walker et al., 2008), and most recently IL-6 acting through its soluble receptor (McGregor et al., 2019). We have also used this method to determine whether responses to cytokines are modified in mice with a cell-specific deletion of gp130, the common receptor used by these cytokines (Johnson et al., 2014). We provide here a full description of how to carry out the in vivo protocol, and how to embed and section tissues using undecalcified histology techniques; micro-computed tomography can also be used for assessment, but this does not allow measurement of bone formation rate using calcein labels. An abbreviated form of this method using single calvarial injections or two days of calvarial injections can also be used to assess gene responses elicited by cytokines in vivo by Western blot (Walker et al., unpublished) and effects on protein expression by immunohistochemistry (Walker et al., 2010).

Materials and Reagents

  1. Plastic flat-bottomed box
  2. Plastic zip-loc bag
  3. Hammer
  4. Thick rubber gloves
  5. Glass bottles for reagents–the methyl-methacrylate will dissolve plastic
  6. Microscope slides (Hurst scientific catalog number: 7107 )
  7. Coverslips (Hurst scientific catalog number: CG12450 )
  8. Glass Scintillation Vials (PerkinElmer catalog number: 6000097 )–methyl-methacrylate will dissolve plastic
  9. Grinding paper (smooth) CarbiMet S SiC P400 (ThermoFisher, catalog number: 16080320 )
  10. Grinding paper (rough) CarbiMet S, 60 P60 (ThermoFisher, catalog number: 16080060 )
  11. Insulin syringe (29 G BD Ultra-fineTM Clifford Hallam, catalog number: 1323684 )
  12. Microfuge tubes 1.5 ml (Pacific labs, Axygen catalog number: MCT175CI )
  13. 6-week old C57BL/6 mice (10 mice per group)
  14. Positive Control: Recombinant mouse Oncostatin M (OSM) (R&D Systems catalog number: 495-MO-025 ) with 2% heat-inactivated mouse serum (made in house)
  15. Your preferred agents/treatments for testing
  16. Phosphate buffered saline (Sigma-Aldrich, catalog number: D8537 )
  17. Isoflurane inhalant anaesthetic (FORTHANE®, AbbVie)
  18. Ethanol
  19. Calcein (Sigma-Aldrich, catalog number: C0875-5G )
  20. Sodium Bicarbonate (Sigma-Aldrich, catalog number: S6014 )
  21. Sodium hydroxide (NaOH)
  22. Acetone
  23. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127-500 g )
  24. Methyl methacrylate (Sigma-Aldrich, catalog number: M55909-500 ml )
  25. Destabilised methylmethacrylate (dMMA)
  26. Dibutyl Phthalate (Merck, catalog number: 800919 )
  27. Granular calcium choride anhydrous (Sigma-Aldrich, catalog number: C-1016 )
  28. Chromium (III) potassium sulphate (Sigma-Aldrich, catalog number: 243361 )
  29. Gelatin from bovine skin (Sigma-Aldrich, catalog number: G9391 )
  30. Butanol (Merck, catalog number: 8222641000 )
  31. Toluene (Crown Scientific, catalog number: 2867322 )
  32. Depex mountant (Crown Scientific, catalog number: 360294H )
  33. Tris (Astral Scientific, catalog number: BIO3094T)
  34. Xylenol orange (Sigma-Aldrich, catalog number: X3500 )
  35. Xylenol Orange Stain (see Recipes)

Equipment

  1. 100 ml beaker
  2. 2 L glass separating funnel
  3. Fume hood
  4. Incubator
  5. Microwave oven
  6. Grinder/Polisher (Buehler Phoenix beta, or equivalent)
  7. Automated microtome, RM2265 Leica, equipped with tungsten carbide blade, or equivalent microtome
  8. Fluorescence microscope equipped with Osteomeasure image analysis system (or equivalent), (Osteometrics, https://www.osteometrics.com/)

Software

  1. Osteomeasure histomorphometry system (Osteometrics, https://www.osteometrics.com/), or equivalent

Procedure

  1. Preparation of cytokines and animals for study
    1. Gain ethics approval from your Institutional Review Board before commencing
      Order mice and allow them to acclimatize to your facility for 2 weeks prior to the experiment. We use C57BL/6 mice at 6 weeks of age (after acclimatization). We use a minimum of 6 mice per treatment group to detect a difference in calvarial thickness of 10%, based on data from our original publication (Walker et al., 2010).
    2. For labelling active bone formation sites, prepare calcein for injections, as follows (see Figure 1 for times at which calcein is injected):
      A solution of 0.01 g sodium bicarbonate plus 0.02 g calcein is made in 10 ml of 0.9% sterile saline (final concentration 2 mg/ml). This solution is injected intraperitoneally or subcutaneously using Insulin syringes (29 G needles) at 0.1ml per 10 g body weight (for a dose of 20 mg/kg). Calcein can be stored in frozen aliquots ready for thawing at the time of use.


      Figure 1. Example of the treatment protocol, showing days of calcein and cytokine injections and tissue collection (end)

    3. Prepare 25 µl aliquots of agents to be tested by calvarial injection
      We recommend making up your agent into 25 µl aliquots (larger and smaller volumes have not been tested). As an example, we use 0.2 µg murine Oncostatin M in a volume of 25 µl for 5 consecutive days as a positive control for all our experiments, including 2% heat-inactivated serum as a carrier. Before commencing we make up sufficient aliquots for the full experiment and freeze 5 aliquots with sufficient for all mice in the treatment group ready for each day of injections.

  2. Injection protocol
    1. Cytokine injections
      Figure 1 shows a time course of the injection protocol. On days 1 to 5 (e.g., Monday to Friday), anaesthetise mouse with 2% isoflurane and 1 L/min oxygen (or anaesthetic as approved by your Institutional Review Board). Using an insulin syringe inject 25 µl cytokine subcutaneous over the skull (between the ears) of the animal (see Figure 2). The subcutaneous injection will form a bubble over the left and right parietal bones.
    2. Calcein injections
      On days 1 and 11, using an insulin syringe inject 0.1 ml per 10 g body weight (i.e., a 20 g mouse will receive 0.2 ml) via intraperitoneal injection. On day 1 this can be done while the animal is still anaesthetized for the calvarial injection.
    3. Before tissue collection
      Make sufficient paraformaldehyde to fix samples (approximately 10 ml per sample). Heat 500 ml PBS to 55 °C (it is quickest to use a microwave oven). Dissolve 2 g paraformaldehyde in the solution, keeping warm on a heater-stirrer. While stirring, add sufficient NaOH to raise pH to 8.0; this is required for the paraformaldehyde to dissolve. Cool to 4 °C before use. Prepare labelled scintillation vials for each animal to be collected.
    4. Collection of tissues for histological analysis
      On day 15, cull mice according to the method approved by your institution. Dissect out the calvaria, using the sagittal and lambdoid suture lines as landmarks (Figure 2A). Place the tissue in 4% paraformaldehyde in PBS for 24 h at 4 °C. On the next day, tip off and dispose of the paraformaldehyde solution and store the samples in 70% ethanol until ready to embed the calvarial tissue in methylmethacrylate.
    5. Preparation of calvarial samples for histological analysis
      When tissue has been fixed, dissect out the back half of the calvariae containing the lambdoid suture to the midpoint of the sagittal suture (see Figure 2B).


      Figure 2. Location of injection site, instructions for dissection and image of the sectioning face of an embedded calvarium. A. Superimposition of calvarial structure over an image of a mouse head showing injection site; B. diagram of a dissected calvarium showing the cutting lines for dissection. C. A calvarial block ready for sectioning. Although we use C57BL/6 mice, we have used an image of a white mouse for clarity.

  3. Preparation of reagents for embedding samples in methyl methacrylate
    1. Destabilization of methyl-methacrylate (dMMA)
      This step is necessary to remove monomethyl hydroquinone, the stabilizing agent in MMA that suppresses polymerisation. Note that all steps using MMA must be conducted in a fume hood, and since MMA will dissolve plastic, all steps need to be conducted using glass vials, funnels, and bottles. All vials, funnels, and bottles must be dry, and MMA should be filtered through granular calcium chloride before use to remove water which will disrupt the embedding process.
      1. Make up 1 L of 0.5% sodium hydroxide (NaOH) by dissolving 0.5 g NaOH in 1 L of distilled water.
      2. Place a large separating funnel into a retort stand in a fume hood.
      3. Pour approximately 500 ml MMA into the large separating funnel.
      4. Add approximately 300 ml 0.5% NaOH to the MMA in the separating funnel.
      5. Shake the separating funnel vigorously for at least 30 sec to mix the NaOH and MMA thoroughly. 
      6. Place in retort stand and allow the layers to settle.
      7. When layers have settled, and while the separating funnel is still in the retort stand, use the tap to drain off the lower layer (NaOH) into a beaker. Discard the NaOH according to your Institution’s protocol for waste disposal (we dispose in the sink with ample tap water).
      8. Repeat Steps C1d to C1g twice; this will use up all the 5% NaOH you made.
      9. Then repeat Steps C1d to C1g three times, using 500 ml distilled water each time, instead of NaOH (water will also settle to the bottom layer).
      10. Finally, place about a 100 ml beaker full of calcium chloride into a folded filter paper in large glass funnel on the retort stand. Place a clean bottle for the destabilized MMA (dMMA) under it.
      11. Slowly transfer the dMMA from the separating funnel through the calcium chloride into the clean bottle. The calcium chloride will remove all traces of water.
      12. Add a small amount of calcium chloride to the clean dMMA to absorb excess water. Store dMMA at 4 °C.
    2. Make up graded acetones
      Make up at least 250 ml each of 70% acetone in water, and 90% acetone in water.
    3. Make up infiltrating solution (total of at least 5 ml per sample)
      Working in a fume hood, mix 90% dMMA with 10% dibutylphthalate (DBP) and 0.05% benzoyl peroxide (BPO). For example, for 10 samples, make up approximately 70 ml, using 63 ml dMMA, 7ml DBP and 0.35 g BPO. Stir until BPO is dissolved, and filter through filter paper containing calcium chloride in a glass funnel to remove any water. Store at 4 °C indefinitely.
    4. Make embedding solution (approximately 20 ml per sample)
      Mix 85% dMMA with 15% dibutylphthalate (DBP) and 4% benzoyl peroxide (BPO). For example, for 10 samples, make up 200 ml, using 170 ml dMMA, 30 ml DBP and 8 g BPO. Stir until BPO is dissolved, and filter through filter paper containing calcium chloride in a glass funnel to remove any water. Store at 4 °C maximum of 1 week.
    5. Make bases for embedding samples. In clean glass scintillation vials, add 2.5 ml embedding solution. To reduce waste, you can make this up using previously used infiltration solution for the bases. Place these, spaced apart from each other in a 37 °C incubator until set.

  4. Infiltrating the calvarial samples in methyl methacrylate:
    Note: Samples should be sheltered from light when not being handled.
    1. Dehydrate samples through graded acetones
      1. Tip off 70% ethanol from all samples, and replace with 70% acetone, store at 4 °C for at least 1 h (up to overnight)
      2. Tip off 70% acetone from all samples, and replace with 90% acetone, store at 4 °C for at least 1 h (up to overnight)
      3. Tip off 90% acetone from all samples, and replace with 100% acetone, store at 4 °C for at least 1 h (up to overnight)
      4. Tip off 100% acetone from all samples, and replace with fresh 100% acetone, store at 4 °C for at least 1 h (up to overnight)
    2. Filter embedding solution
      While samples are in 100% acetone, allow embedding solution (made in Step C3) to come to room temperature in a fume hood. When at room temperature, working in the fume hood, filter the solution through filter paper containing calcium chloride in a glass funnel to remove any water.
    3. Infiltrate samples
      Working in the fume hood, tip 100% acetone off all samples and replace it with approximately 5 ml embedding solution. Store at 4 °C in the dark for at least 3 nights before embedding.

  5. Embedding the calvarial samples in methyl methacrylate
    1. When bases have set, you can embed the calvarial samples.
    2. Allow samples, embedding solution, and bases to come to room temperature in a fume hood.
    3. In a fume hood, place 15 ml fresh embedding solution on enough bases for all the calvarial samples you need to embed. Allow the bases to soften by leaving them at room temperature for about 20-30 min.
    4. In the fume hood, when base has softened, transfer bone and sample label to vials containing bases and embedding solution. Place the calvaria close to the side of the vial and orient the samples in the base with the superior side of the calvariae up. Used infiltration solution can be stored at 4 °C and used to make bases for future samples (Step C5).
    5. Place vials in a plastic flat-bottomed box with sufficient water to reach the same level as the MMA in the vials to distribute generated heat. Be sure to space vials at least 5 mm apart so that heat can dissipate. Leave at 37 °C overnight or until plastic has set.
    6. When plastic has set, place samples in freezer for 1 h.
    7. To remove glass vials from samples, place each scintillation vial into a sealed plastic bag and hit with a hammer to break the glass. Wearing thick gloves to protect fingers, remove the plastic embedded bone from the broken glass vial.

  6. Preparing Fol’s coated slides
    1. In a 500 ml bottle, dissolve 6 g gelatin in 288 ml dH2O at 60 °C; heat the water quickly in a microwave oven.
    2. Make up solution of 4 g chromium potassium sulphate in 20 ml dH2O.
    3. Once gelatin is cool, add 120 ml 95% ethanol.
    4. Then add dissolved chromium potassium sulphate.
    5. To coat rack up slides in a slide holder/rack and dip for 30 sec per rack.
    6. Dry overnight but leave for 2 nights if placing them in boxes for use (otherwise the slides stick together).

  7. Prepare Xylenol Orange Stain (see Recipes)

  8. Sectioning calvarial samples
    1. On the grinder-polisher, using P60 grinding paper, grind the sample down until you have reached the surface of the bone to be sectioned. Then smooth it over with the 320 grinder paper. Grind the top and bottom of the sample so that it can be held in your microtome’s sample holder, and grind the edges for sectioning (see Figure 2C).
    2. On the microtome, using the tungsten carbide blade with 70% ethanol to float the samples, take 5-10 micron coronal sections from the midpoint of the sagittal suture and place on Fols coated slides. Once section is on the slide, use a drop of 95% ethanol to make the sample more flexible and gently stretch it along the slide to remove folds. Place a small piece of bagging plastic on top of the section, and a clean dry microscope slide on top of that. Clamp sections together (with a large bulldog clip) and bake overnight at 37 °C.
    3. The next day, remove sections from clamps, stain with xylenol orange, dehydrate, and coverslip with DePex mountant.

  9. Staining calvarial samples
    While it is possible to analyse the sections without stain, the Xylenol Orange stain provides a good contrast and makes it easier to see the samples.
    1. Deplasticise in two changes of cellosolve acetate for 25 min each.
    2. Rehydrate in graded ethanols, as follows:
      100% x 3 min
      1 00% x 3 min
      80% x 3 min
      60% x 3 min
      Tap water x 5 min
    3. Stain sections in xylenol orange for 3 min.
    4. Rinse in dH2O.
    5. Blot both sides of samples thoroughly.
    6. Pass sections through the following solutions in order:
      Butanol x 2
      1:1 Butanol/Toluene x 1
      Toluene x 2
    7. Coverslip with DePex.
    8. Allow to dry, sheltered from light.

  10. Measuring calvarial thickness and bone formation rates, using Osteomeasure or similar image analysis software
    1. Measure coverslipped tissue sections with a 20x objective. Measure the total calvarial thickness and both single and double labelled surface on the superior side of the calvaria (the injected side). Do not measure the central 800 microns containing the sagittal suture. Measure 8-12 fields on both sides of the sagittal suture.
    2. Download bone thickness, double labelled surface, bone surface, and single labelled surface, and calculate mineral appositional rate and calvarial thickness. Since the entire surface is usually covered with double label, we usually do not calculate bone formation rate, but if your agent reduces bone formation this may be important. If any samples completely lack double calcein labelling throughout the sample, they should be excluded from analysis as this likely reflects a lack of calcein injection.


      Figure 3. Imaging and measurement of calvarial sample. A. Composite image of a single calvarial section stained with Xylenol Orange showing the calcein labels; scale bar = 50 μm. Yellow arrows show approximate region of measurement that would be used for this sample. B. Example of a sample being measured, showing measurement of double labels on the superior surface of a calvarial section.

Data analysis

Compare calvarial thickness and mineral apposition rate data between groups by one-way ANOVA to compare multiple treatment groups.

Recipes

  1. Xylenol Orange Stain
    1. Make up 0.5 M Tris buffer: dissolve 6.06 g Tris in 100 ml distilled water
    2. Add 1 g Xylenol Orange
    3. Adjust pH to 9.0
    4. Store in a sealed bottle at room temperature indefinitely

Acknowledgments

The authors thank the staff of the St. Vincent’s Health Bioresources Centre for excellent animal care and assistance, and Joshua Johnson who helped in developing methods for analysis. This work was supported by NHMRC Grants 1120978 and 1058625. NAS is supported by an NHMRC Senior Research Fellowship. St Vincent’s Institute acknowledges the support of the Victorian State Government OIS program. This protocol was based on an initial study by Cornish et al. (1993).

Competing interests

The authors have no financial or non-financial competing interests.

Ethics

This protocol was approved by the St. Vincent’s Health Melbourne Animal Ethics Committee AEC#005/16 valid May 2016, December 2019.

References

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  2. Blank, M. and Sims, N. A. (2019). Cellular processes by which osteoblasts and osteocytes control bone mineral deposition and maturation revealed by stage-specific EphrinB2 knockdown. Curr Osteoporos Rep 17(5):270-280.
  3. Cornish, J., Callon, K., King, A., Edgar, S. and Reid, I. R. (1993). The effect of leukemia inhibitory factor on bone in vivo. Endocrinology 132(3): 1359-1366.
  4. Dallas, S. L. and L. F. Bonewald (2010). Dynamics of the transition from osteoblast to osteocyte. Ann N Y Acad Sci 1192: 437-443.
  5. Falconi, D. and Aubin, J. E. (2007). LIF inhibits osteoblast differentiation at least in part by regulation of HAS2 and its product hyaluronan. J Bone Miner Res 22(8): 1289-1300.
  6. Johnson, R. W., Brennan, H. J., Vrahnas, C., Poulton, I. J., McGregor, N. E., Standal, T., Walker, E. C., Koh, T. T., Nguyen, H., Walsh, N. C., Forwood, M. R., Martin, T. J. and Sims, N. A. (2014). The primary function of gp130 signaling in osteoblasts is to maintain bone formation and strength, rather than promote osteoclast formation. J Bone Miner Res 29(6): 1492-1505.
  7. Malaval, L. and Aubin, J. E. (2001). Biphasic effects of leukemia inhibitory factor on osteoblastic differentiation. J Cell Biochem Suppl Suppl 36: 63-70.
  8. Malaval, L., Gupta, A. K. and Aubin, J. E. (1995). Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures. Endocrinology 136(4): 1411-1418.
  9. Malaval, L., Liu, F., Vernallis, A. B. and Aubin, J. E. (2005). GP130/OSMR is the only LIF/IL-6 family receptor complex to promote osteoblast differentiation of calvaria progenitors. J Cell Physiol 204(2): 585-593.
  10. McGregor, N. E., Murat, M., Elango, J., Poulton, I. J., Walker, E. C., Crimeen-Irwin, B., Ho, P. W. M., Gooi, J. H., Martin, T. J. and Sims, N. A. (2019). IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis. J Biol Chem 294(19): 7850-7863.
  11. McGregor, N. E., Poulton, I. J., Walker, E. C., Pompolo, S., Quinn, J. M., Martin, T. J. and Sims, N. A. (2010). Ciliary neurotrophic factor inhibits bone formation and plays a sex-specific role in bone growth and remodeling. Calcif Tissue Int 86(3): 261-270.
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  13. Orriss, I. R., Hajjawi, M. O. R., Huesa, C., MacRae, V. E. and Arnett, T. R. (2014). Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats. International journal of molecular medicine 34(5): 1201-1208.
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  16. Walker, E. C., McGregor, N. E., Poulton, I. J., Pompolo, S., Allan, E. H., Quinn, J. M., Gillespie, M. T., Martin, T. J. and Sims, N. A. (2008). Cardiotrophin-1 is an osteoclast-derived stimulus of bone formation required for normal bone remodeling. J Bone Miner Res 23(12): 2025-2032.
  17. Walker, E. C., McGregor, N. E., Poulton, I. J., Solano, M., Pompolo, S., Fernandes, T. J., Constable, M. J., Nicholson, G. C., Zhang, J. G., Nicola, N. A., Gillespie, M. T., Martin, T. J. and Sims, N. A. (2010). Oncostatin M promotes bone formation independently of resorption when signaling through leukemia inhibitory factor receptor in mice. J Clin Invest 120(2): 582-592.
  18. Walker, E. C., Truong, K., McGregor, N. E., Poulton, I. J., Martin, T. J. and Sims, N. A. Cortical bone consolidation requires local suppression of cortical osteoclasts through gp130/STAT3 signalling in osteocytes by SOCS3. (unpublished)

简介


[摘要 ] 骨骼形成发生在胚胎发生,骨骼生长以及整个生命的骨骼更新过程中。在骨形成过程中,成骨细胞沉积了一种含胶原蛋白的基质(称为类骨质),该基质通过掺入矿物质晶体而逐渐硬化。尽管可以诱导成骨细胞分化并在培养物中沉积矿物质,但该系统并不总是提供反映试剂在体内刺激骨骼形成能力的结果。该协议描述了一种快速可靠的方法,用于测试体内局部骨形成剂的局部给药。 在这种方法中,连续5天给小鼠注射相关药物。在用于测试的试剂之前和之后注射荧光染料标记,并通过未脱钙的骨组织学和组织形态计量术收集并分析样品。这提供了一种评估试剂刺激骨形成能力的可靠方法,如果使用短期修饰,也可以用于测试骨中对相同刺激的基因反应。

[背景 ] 骨形成是为骨架的发展所需的基本过程,对于骨骼生长的进展,并保持整个生命骨结构作为骨架不断重塑。骨形成是通过三个步骤发生的过程:(1)富含胶原的类骨质沉积,(2)通过在胶原纤维周围沉积生物磷灰石晶体而发生的类骨矿化,以及(3)矿化的骨质成熟(Blank and Sims ,2019)。这三个步骤由源自共同祖细胞的两种细胞类型介导。成骨细胞沉积类骨质并开始矿化,并且随着类骨质的沉积,一些成骨细胞被包埋在骨基质内并分化为骨细胞(Dallas and Bonewald ,2010)。这些嵌入的细胞还控制骨骼矿化的程度和性质(Vrahnas 等,2019)。

鉴定可刺激骨形成的药物是用于理解骨架的生物学和用于开发可用于促进在诸如骨质疏松症,成骨不全,骨脆弱的条件骨形成新剂临界骨软化,以及开发的药剂在外科手术中可以促进骨折愈合或刺激骨骼形成。通常,测试此类药剂的最简单系统是使用原代培养的成骨细胞(Orriss 等,2014),或区分基质细胞系,例如MC-3T3-E1 (Quarles 等,1992)和Kusa 4b10细胞( Allan et al。,2003)在成骨细胞培养基中。这些系统通常用于通过评估各种药物对成骨细胞标志物基因的mRNA水平,碱性磷酸酶活性和矿物质沉积的影响来研究成骨细胞的分化(Allan 等,2003; McGregor 等,2010; Walker 等。,2010)。但是,这些系统并不总是反映体内反应。一个明显的例子是,体内早期研究表明白血病抑制因子可刺激骨形成(Metcalf和Gearing ,1989 ;Cornish 等,1993),而体外实验中白血病抑制因子既可刺激也可抑制成骨细胞分化(Malaval 等,1995; Malaval和Aubin,2001; Malaval 等,2005; Falconi和Aubin,2007)。

在该协议中,我们描述了颅盖注射模型的使用,该模型可以测试药物刺激体内骨骼形成的能力。这种方法只需要少量的刺激剂,最初由Cornish (Cornish et al。,1993)描述,以解决关于白血病抑制因子是否能促进骨骼形成的争议。我们已经将康沃尔方法改编为包括钙黄绿素标记以测量骨形成,并且我们已经利用它来测试一系列细胞因子,包括癌抑素M (Walker 等,2010),心肌营养素1 (Walker 等。,2008),并且最近IL-6通过其可溶性受体起作用(McGregor 等,2019)。我们还已经使用该方法来确定在具有细胞特异性缺失gp130的小鼠中对细胞因子的应答是否被修饰,gp130是这些细胞因子所使用的共同受体(Johnson 等,2014)。我们在这里提供有关如何执行体内实验方案的完整说明,以及如何使用未脱钙的组织学技术将组织包埋和切片。微型计算机断层扫描也可以用于评估,但这无法使用钙黄绿素标记物测量骨形成率。使用单次颅盖注射或两天颅盖注射的该方法的缩写形式,还可用于通过Western印迹评估细胞因子在体内引起的基因反应(Walker 等,未发表),以及通过免疫组织化学对蛋白质表达的影响(Walker 等)。等,2010)。

关键字:成骨细胞, 骨细胞, 骨形成, 细胞因子, 生长因子

材料和试剂


 


塑料平底盒
塑料拉链袋
锤子
厚橡胶手套
试剂玻璃瓶-甲基丙烯酸甲酯会溶解塑料
显微镜载玻片(赫斯特科学目录号:7107)
盖玻片(赫斯特科学目录号:CG12450)
玻璃闪烁瓶(PerkinElmer目录编号:6000097)–甲基丙烯酸甲酯会溶解塑料
砂纸(光滑)CarbiMet S SiC P400(ThermoFisher ,目录号:16080320)
粗砂纸CarbiMet S,60 P60(ThermoFisher ,目录号:16080060)
胰岛素注射器(29 G BD Ultra- fine TM Clifford Hallam,货号:1323684)
1.5 ml 微量离心管(太平洋实验室,Axygen 目录号:MCT175CI)
6 - 周龄的C57BL / 6小鼠(每组10只小鼠)
阳性对照:重组小鼠间接成本ATIN M(OSM)(R&d系统目录号:495-MO-025),用2%热失活的鼠血清(在自制)
您首选的测试剂/治疗
磷酸盐缓冲盐水(Sigma-Aldrich ,目录号:D8537 )
异氟烷吸入麻醉(FORTHANE ® ,艾伯维)
乙醇
钙黄绿素(Sigma-Aldrich公司,目录号:C0875-5G)
碳酸氢钠(Sigma-Aldrich,目录号:S6014)
氢氧化钠(NaOH)
丙酮
多聚甲醛(Sigma-Aldrich,目录号:158127-500 g)
甲基丙烯酸甲酯(Sigma-Aldrich,目录号:M55909-500 ml)
不稳定的甲基丙烯酸甲酯(dMMA )
邻苯二甲酸二丁酯(默克,目录号:800919)
粒状钙氯化物的无水(Sigma-Aldrich公司,目录号:C-1016)
硫酸铬(III)(Sigma-Aldrich ,目录号243361)
牛皮肤中的明胶(Sigma-Aldrich ,目录号:G9391)
丁醇(默克,目录号:8222641000)
甲苯(Crown Scientific,目录号:2867322)
Depex 封固剂(Crown Scientific,目录号:360294H)
Tris (Astral Scientific ,目录号:BIO3094T )
二甲酚橙(Sigma-Aldrich ,目录号:X3500)
二甲苯酚橙染色剂(见配方)
 


设备


 


100毫升烧杯
2 L玻璃分液漏斗
通风柜
孵化器
微波炉
研磨机/抛光机(Buehler Phoenix beta,或同等水平)
配备了碳化钨刀片的自动切片机,RM2265 Leica,或等效的切片机
配备了Osteomeasure 图像分析系统(或等效工具)的荧光显微镜,(Osteometrics ,https : //www.osteometrics.com/ )
 


软件


 


骨测量组织形态测量系统(Osteometrics ,https : //www.osteometrics.com/ )或同等学历
程序


 


准备用于研究的细胞因子和动物
开始之前先获得机构审查委员会的道德认证
订购小鼠并让它们在实验前2周适应您的设备。我们使用6周龄(适应后)的C57BL / 6小鼠。根据我们原始出版物的数据(Walker 等,2010),我们每个治疗组至少使用6只小鼠来检测颅骨厚度差异10%。


为了标记活跃的骨形成部位,请准备注射用钙黄绿素,如下所示(关于钙黄绿素的注射时间,请参见图1 ):
的溶液0.01 G钠碳酸氢盐加0.02 克Ç alcein 在10制成毫升0.9%无菌盐水(终浓度2 毫克/毫升)。使用胰岛素注射器(29 G针)腹膜内或皮下注射该溶液,剂量为每10 克体重0.1 毫升(剂量为20 毫克/千克)。钙黄绿素可以以冷冻等分试样形式储存,以备使用时解冻。             


 


D:\ Reformatting \ 2020-1-6 \ 1902891--1316 Natalie Sims 781881 \ Figs jpg \图1.jpg


图1. 治疗方案示例,显示了钙黄绿素和细胞因子注射的天数以及组织收集(结束)


 


准备要通过颅盖静脉注射测试的试剂的25 µl等分试样
我们建议将您的试剂分成25微升等分试样(较大和较小的体积尚未经过测试)。例如,我们连续5天以25 µl的体积使用0.2 µg鼠Onostatin M作为我们所有实验的阳性对照,包括2%热灭活的血清作为载体。开始之前,我们为整个实验准备了足够的等分试样,并冷冻了5个等分试样,足够治疗组中的所有小鼠准备每天注射。


 


注射方案
细胞因子注射
图1显示了注射方案的时间过程。在1〜5天(例如,星期一至星期五),anaesthetise 鼠标2%我soflurane和1升/分钟Ø xygen(或麻醉核准您的机构审查委员会)。使用胰岛素注射器在动物的头骨(耳朵之间)皮下注射25 µl细胞因子(见图2)。皮下注射会在左右顶骨上形成气泡。


钙黄绿素注射液
在第1天和第11天,使用胰岛素注射器通过腹膜内注射,每10 g体重注射0.1 ml (即20 g小鼠将接受0.2 ml)。在第1天,可以在仍然麻醉动物进行颅骨注射的同时进行此操作。


收集组织之前
使足够的多聚甲醛能够固定样品(每个样品约10 ml)。将500 ml PBS 加热至55   °C(使用微波炉最快)。将2 g多聚甲醛溶解在溶液中,并在加热器上保持温暖。在搅拌的同时,添加足够的NaOH以将pH升至8.0;这是溶解多聚甲醛所必需的。使用前冷却至4 °C 。准备要收集的每个动物的标记闪烁瓶。


收集胫骨以进行组织学分析
在第15天,根据您所在机构批准的方法对小鼠进行剔除。解剖出颅盖,使用矢状和lambdoid缝合线作为界标(图2A)。放置在PBS中的4%多聚甲醛的组织24 在4小时℃下。第二天,放倒并处理低聚甲醛溶液,并将样品存储在70%的乙醇中,直到准备将颅盖组织嵌入甲基丙烯酸甲酯中。


制备颅骨SA mples进行组织学分析
当组织已经被固定的,解剖出含有人字缝到矢状缝的中点(在颅盖骨的后半部小号EE图2B)。


 


D:\ Reformatting \ 2020-1-6 \ 1902891--1316 Natalie Sims 781881 \ Figs jpg \图2.jpg


图2 。注射部位的位置,解剖说明和嵌入式颅骨切面的图像。A. 在显示注射部位的小鼠头部的图像上叠加颅盖结构;B.解剖的颅骨图,显示解剖的切割线。C. 准备好颅骨节段。尽管我们使用C57BL / 6小鼠,但为了清楚起见,我们使用了白色小鼠的图像。


 


用于将样品包埋在甲基丙烯酸甲酯中的试剂的制备
Destabilizatio 甲基-甲基丙烯酸酯的N(DMMA )
该步骤对于去除单甲基氢醌是必要的,该单甲基氢醌是MMA中抑制聚合的稳定剂。请注意,所有使用MMA的步骤都必须在通风橱中进行,并且由于MMA会溶解塑料,因此所有步骤都需要使用玻璃小瓶,漏斗和瓶子进行。所有小瓶,漏斗和瓶子都必须干燥,并且在使用MMA之前应先通过粒状氯化钙过滤以除去会破坏包埋过程的水。


通过将0.5 g NaOH 溶解在1 L 的蒸馏水中制成1 L 的0.5%氢氧化钠(NaOH)。
将一个大的分液漏斗放入通风橱中的蒸馏塔中。
将约500毫升MMA倒入大的分液漏斗中。
在分液漏斗中向MMA中添加约300 ml 0.5%NaOH。
剧烈摇晃分液漏斗至少30秒钟,以使NaOH和MMA充分混合。
放置在脱水缸中,并允许各层沉降。
待各层沉降后,并且分液漏斗仍在脱水缸中时,请使用水龙头将下层(NaOH)排入烧杯中。根据您所在机构的协议处理NaOH,将其丢弃(我们将使用大量自来水将其丢弃在水槽中)。
重复小号TEPS C1 d 到C1 克两次; 这将用尽您制成的所有5%NaOH。
然后重复小号TEPS C1D到C1G三次,每次用500毫升蒸馏水的水,而不是将NaOH(水也将沉淀到底层)。
最后,将约100 毫升装满氯化钙的烧杯放入折叠的滤纸中,该滤纸放在re架上的大玻璃漏斗中。在其下方放置一个干净的瓶,以放置不稳定的MMA(d MMA )。
将dMMA 从分液漏斗通过氯化钙缓慢转移到干净的瓶子中。氯化钙将去除所有痕量的水。
向干净的dMMA中添加少量氯化钙,以吸收过量的水。将dMMA 储存在4°C。
组成分级丙酮
分别溶解至少250毫升的70%丙酮和90%丙酮。


配制浸润液(每个样品至少5 毫升)
在通风橱中工作,将90%dMMA 与10%邻苯二甲酸二丁酯(DBP)和0.05%过氧化苯甲酰(BPO)混合。例如,对于10个样品,使用63 ml dMMA ,7 ml DBP和0.35 g BPO 大约补足70 ml 。搅拌直至BPO溶解,然后在玻璃漏斗中通过含氯化钙的滤纸过滤以除去所有水。无限期储存在4 °C。                                                                                   


制作包埋溶液(每个样品约20 ml)
将85%的dMMA 与15%的邻苯二甲酸二丁酯(DBP)和4%的过氧化苯甲酰(BPO)混合。例如,对于10个样品,使用170 ml dMMA ,30 ml DBP和8 g BPO 补足200 ml 。搅拌直至BPO溶解,然后在玻璃漏斗中通过含氯化钙的滤纸过滤以除去所有水。储存在4 °C下最多1周。                                                                                   


为嵌入样本奠定基础。在干净的玻璃闪烁瓶中,加入2.5 ml包埋溶液。为了减少浪费,您可以使用以前使用的渗透解决方案来弥补这一问题。将彼此隔开一定距离的这些艺术品放在37°C的培养箱中直至凝固。
 


将颅骨样品浸入甲基丙烯酸甲酯中:
注:小号amples应避风亮时不被处理。


通过分级丙酮对样品进行脱水
倾倒所有样品中的70%乙醇,并用70%丙酮代替,在4 °C下储存至少1 h(最多过夜)
从所有样品中取出70%的丙酮,并用90%的丙酮代替,在4 °C下保存至少1小时(最多过夜)。
从所有样品中取出90%的丙酮,并用100%的丙酮代替,在4 °C下保存至少1小时(最多过夜)。
提示关闭从所有样品100%丙酮,并用新鲜的100%更换一个cetone,储存在4 ℃下至少1个小时(最多过夜)
过滤器嵌入解决方案
当样品置于100%丙酮中时,让包埋溶液(步骤C3中制得)在通风橱中达到室温。在室温下在通风橱中工作时,将溶液通过玻璃漏斗中的含氯化钙滤纸过滤,以除去任何水分。


渗透样品
在通风橱中操作,从所有样品中倒出100%丙酮,并用约5 ml包埋溶液替换。嵌入之前,请在4 °C 的黑暗环境中存放至少3晚。


 


将颅骨样本嵌入甲基丙烯酸甲酯中
设置基准后,可以嵌入颅骨样本。
使样品,包埋溶液和碱液在通风橱中达到室温。
在通风橱中,将15 ml新鲜的包埋溶液放在足以包埋所有颅盖样品的基底上。将其在室温下放置约20-30分钟,以使其变软。
在通风橱中,当底座变软时,将骨骼和样品标签转移到装有底座和包埋溶液的小瓶中。将颅盖放置在靠近小瓶的一侧,然后将样品放在颅盖的上侧向上的底部。用过的渗透溶液可以储存在4 °C下,并为以后的样品打基础(步骤C5)。
将小瓶放入塑料制的平底盒子中,并用水充分,使其达到与小瓶中MMA 相同的水平,以散发产生的热量。确保将小瓶间隔至少5毫米,以便散发热量。在37 °C下放置过夜或直到塑料凝固。
凝固塑料后,将样品放在油炸锅中1小时。
要从样品瓶中取出玻璃瓶,请将每个闪烁瓶放入密封的塑料袋中,并用锤子将玻璃瓶打碎。戴着厚手套以保护手指,从破碎的玻璃小瓶中取出嵌入的塑料骨头。
 


准备Fol的涂层幻灯片
1. 在500 °C的瓶子中,在60 °C下,将6 g明胶溶解在288 ml dH 2 O中;在微波炉中快速加热水。      


2. 配制4 g硫酸铬钾在20 ml dH 2 O中的溶液。      


3. 明胶冷却后,加入120 ml 95%乙醇。      


4. 然后添加溶解的硫酸铬钾。      


5. 要在载玻片固定器/机架中对载玻片进行涂层处理,每个载玻片浸入30秒。      


6. 干燥过夜,但如果将其放在盒子中待用则放置2晚(否则,幻灯片会粘在一起)。      


 


准备二甲酚橙站中(见食谱)
 


切片颅骨样本
在研磨抛光机上,使用P60砂纸向下研磨样品,直到到达要切骨的表面为止。然后用320研磨纸将其弄平。研磨样品的顶部和底部,以便可以将其固定在切片机的样品架中,然后研磨边缘以进行切片(请参见图2 C)。
在切片机上,使用带有70%乙醇的碳化钨刀片使样品漂浮,从矢状缝线的中点截取5-10微米的标准切片并将其放在Fols 涂层载玻片上。将切片放在载玻片上后,使用一滴95%的乙醇使样品更具柔韧性,并沿载玻片轻轻拉伸以除去褶皱。将一小袋装袋塑料放在该部分的顶部,然后将一块干净的干燥显微镜载玻片放在该部分的顶部。将各部分夹在一起(用大的牛头犬夹子),并在37 °C下烘烤过夜。
第二天,从夹具上取下切片,用二甲酚橙染色,脱水,并用DePex 封固剂盖玻片。
 


颅骨样本染色
虽然可以分析无污点的切片,但是二甲酚橙的污点可以提供良好的对比度,并且更易于观察样品。


两次更换醋酸纤维溶纤剂,分别进行25分钟的去塑作用。
在分级乙醇中重新水合,如下所示:
100%x 3分钟


1 00%x 3分钟             


80%x 3分钟


60%x 3分钟


                                                                      自来水x 5分钟             


用二甲酚橙将切片染色3分钟。
用dH 2 O 冲洗。
将样品的两面彻底吸干。
按以下顺序依次通过以下解决方案:
丁醇x 2


1:1丁醇/甲苯x 1


甲苯x 2


带DePex的Coverslip 。
使其干燥,避光。
 


使用Osteomeasure 或类似的图像分析软件测量颅骨厚度和骨形成率
测量盖玻片p 编组织切片用20倍物镜。测量总颅盖厚度以及颅盖上侧(注射侧)的单面和双面标记表面。不要测量包含矢状缝线的中心800微米。测量矢状缝合线两侧的8-12个视野。
做wnload骨厚度,双标记表面,骨表面,和单标记表面,并计算矿化沉积速率和颅骨厚度。由于通常整个表面都覆盖有双标签,因此我们通常不计算骨形成率,但是如果您的药剂减少了骨形成,这可能很重要。如果任何样品在整个样品中完全缺乏双钙黄绿素标记,则应将它们从分析中排除,因为这很可能反映出缺乏钙黄绿素注射液。
 


D:\ Reformatting \ 2020-1-6 \ 1902891--1316 Natalie Sims 781881 \ Figs jpg \图3.jpg


图3 。颅骨样本的成像与测量。A. 用二甲苯酚橙染色的单个颅盖部分的复合图像,显示了钙黄绿素标记;比例尺= 50 μ米。黄色箭头显示将用于此样本的近似测量区域。B.被测样品的例子,显示了在颅盖部分上表面的双标记的测量。


 


数据分析


 


比较颅骨厚度和矿物appos 我和灰率数据组之间通过单因素ANOVA来比较多个治疗组。


 


[R ecipes


 


二甲苯酚橙染色
组成0.5 M Tris缓冲液:将6.06 g Tris溶于100 ml蒸馏水
加入1克二甲酚橙                           
调节pH至9.0
无限期在室温下储存在密封瓶中
 


致谢


 


作者感谢圣文森特健康生物资源中心的工作人员提供了出色的动物护理和帮助,并感谢约书亚·约翰逊(Joshua Johnson)帮助开发了分析方法。这项工作得到了NHMRC赠款1120978 和1058625的支持。NAS受NHMRC高级研究奖学金的支持。圣文森特学院感谢维多利亚州政府OIS计划的支持。该协议基于Cornish 等人的初步研究。(1993)。


 


利益争夺


 


作者没有金融或非金融竞争利益。


 


伦理


 


该规程已由圣文森特健康墨尔本动物伦理委员会AEC#005/16 批准,有效期为2016 年5月,2019年12月。


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. McGregor, N. E., Poulton, I. J., Walker, E. C. and Sims, N. A. (2020). Testing Bone Formation Induction by Calvarial Injection Assay in vivo. Bio-protocol 10(6): e3560. DOI: 10.21769/BioProtoc.3560.
  2. McGregor, N. E., Murat, M., Elango, J., Poulton, I. J., Walker, E. C., Crimeen-Irwin, B., Ho, P. W. M., Gooi, J. H., Martin, T. J. and Sims, N. A. (2019). IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis. J Biol Chem 294(19): 7850-7863.
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