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Exogenous (human or mouse) mesenchymal stem cells (MSCs) seed in HA scaffold and transplant subcutaneously in immunodeficient mice, the cells can finally form bone tissues in the in vivo environment. This protocol describes how to get heterotopic bone formation in mice by mesenchymal stem cells (MSCs) in hydroxyapatite (HA) scaffolds. This is a simple and robust approach to detect the bone formation by tissue engineering approaches in vivo, and it also fits for examining the roles of different factors in bone formation.

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In vivo Heterotopic Bone Formation Assay Using Isolated Mouse and Human Mesenchymal Stem Cells
采用分离的小鼠和人间充质干细胞进行体内异位骨形成分析

干细胞 > 成体干细胞 > 间充质干细胞
作者: Li Chen
Li ChenAffiliation: Molecular Endocrinology Laboratory (KMEB), Department of Endocrinology, Odense University Hospital, Odense, Denmark
For correspondence: lchen@health.sdu.dk
Bio-protocol author page: a1968
 and Nicholas Ditzel
Nicholas DitzelAffiliation: Molecular Endocrinology Laboratory (KMEB), Department of Endocrinology, Odense University Hospital, Odense, Denmark
Bio-protocol author page: a1969
Vol 5, Iss 3, 2/5/2015, 3073 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1389

[Abstract] Exogenous (human or mouse) mesenchymal stem cells (MSCs) seed in HA scaffold and transplant subcutaneously in immunodeficient mice, the cells can finally form bone tissues in the in vivo environment. This protocol describes how to get heterotopic bone formation in mice by mesenchymal stem cells (MSCs) in hydroxyapatite (HA) scaffolds. This is a simple and robust approach to detect the bone formation by tissue engineering approaches in vivo, and it also fits for examining the roles of different factors in bone formation.
Keywords: Bone formation(骨形成), Mouse model(小鼠模型), Stem cells(干细胞)

[Abstract]

Materials and Reagents

  1. Human or mouse mesenchymal stem cells (cultured, treated or transfected)
    Note: If using the primary bone marrow stem cells (attached cells from bone marrow culturing), it’s better to use cells in low passages (not over passage 3); if using MSC cell lines, it’s also better to use low passages than high passages.
  2. Mice [NOD.CB17-Prkdc/J mice (NOD/SCID), around 8 weeks old]
  3. Hydroxyapatite/tricalcium phosphate (HA/TCP) (Zimmer, catalog number: 60-130)
  4. 4% paraformaldehyde
  5. Hematoxylin/Eosin (H&E) staining solution
  6. Formic acid solution (see Recipes)

Equipment

  1. 1 ml syringes
  2. Cotton sticks (Pro-Ophtha)
  3. Scalpels
  4. Racks (for fixing the syringes in vertical position in cell incubator)
  5. Tools for mouse surgery (scalpel, forceps, needle holder, scissors, sutures, sterile swabs, sterile drapes)
  6. Scan microscopy (Leica Microsystems or any scan microscopy)
  7. Computer (with image analysis software, such as ImageJ® or Photoshop CS®)

Software

  1. ImageJ® or Photoshop CS®

Procedure

  1. Prepare the implants (stromal stem cells with HA/TCP scaffolds)
    1. Take the 1 ml syringes, cut off the tips with a scalpel and let the syringe tip side open, but keep the piston inside as it is.
    2. Weight 40 mg hydroxyapatite (HA/TCP) and transfer into each syringe.
    3. Put a cut cotton stick to close the open side of the syringe.
    4. Autoclave the syringes with hydroxyapatite (HA/TCP).
    5. Take the sterilized syringes and HA/TCP back to cell culture room. Place syringes in a rack (syringes must be kept vertical).
    6. Open the syringes, add 100 μl 10% FBS MEM medium to each HA/TCP scaffold. Mix by 1 ml tip up and down pipetting to wet the HA/TCP thoroughly.
    7. MSC cells (treated or transfected) are trypsinized and counted.
    8. 5 x 105 cells in 200 μl medium are transferred into HA/TCP in the syringe.
    9. Mix thoroughly but gently by 1 ml tip up and down pipetting.
    10. Place racks with syringes in the incubator, 37 °C, 5% CO2, overnight (see in Figure 1).


      Figure 1. Racks with syringes

    11. Next day, bring the transplant cells in the HA/TCP into the animal room. Ready for transplant cells subcutaneously in mice.

  2. Implantation in mice
    1. NOD/SCID mice, feed in sterilized animal room, around 8 weeks old. Each mouse can hold four implants in four positions (right-front, left-front, right-back, left-back).
      Note: In order to avoid variation induced by individual mice or implantation position, suggest that in each mouse, with the control and experiment group; in each position, randomly implant the different treated samples.
    2. Anaesthesia the mice using the general protocol. Let the mice lay relax with the back upside (see in Figure 2A).
    3. Shaving the fur of mice at middle of back, at one side of spine. Ethanol sterilizes the skin at the shaved area. Let it dry for a while (see in Figure 2B).
    4. Cut a 1-1.5 cm gap in the skin (see in Figure 2C).
    5. Push the piston of the syringe with cells inside, to get rid of the extra medium upper the implant.
    6. Insert the 1 ml syringe with cells in HA/TCP into the cut gap in the skin. Go 1.5 cm forward under the skin to the front position from the cut gap to reach the front implantation position (see in Figure 2D-E).
    7. Push the piston of syringe and let the implant (HA/TCP with cells) out, settle implant at the position under the skin (see in Figure 2E).
    8. Slowly drag the syringe out, discard it (see in Figure 2F).
    9. Repeat the same step using another implant, this time let the syringe insert 1.5 cm back under the skin to the back position from the cut gap to reach the back implantation position.
    10. After both front and back implants done, suture the cut over the skin (see in Figure 2G).
    11. Do the same on the other side of spine on the back of the mouse (see in Figure 2H).


      Figure 2. Implantation in mice

    12. Continue till all the implants put into mice.
    13. Keep the mice warm and under-watch in watch room overnight.
    14. Then transfer mice into the regular feeding room (sterilized animal room).
    15. Wait for 8 weeks till the bone formation in the implants.

  3. Fixing, embedding and H&E staining of implants
    1. Sacrifice the mice, take out each implant from each position, directly fix in 4% paraformaldehyde overnight.
    2. Discard the paraformaldehyde, decalcified implant in formic acid for 3 days at 4 °C, then keep in PBS at 4 °C.
    3. Embed the implants in paraffin using the general protocol.
    4. Cut each implant into sections (4-μm), at least three sections at different three positions of the implant.
    5. Stain all the sections with H&E Y using the general protocol.

  4. Scan the sections under microscopy
    1. Take the section staining slides to the microscopy room.
    2. Scan each section (the whole section) under microscopy.
    3. Save the scanned pictures in disk.

  5. Analyze the bone formation volume by image analysis software
    1. Open each section scan picture in Photoshop or other image analysis software. The bone formation area stains as red (pink), the HA/TCP scaffolds stain as dark brown, non-differentiated MSCs show as blue spots.
    2. Pick all the red areas (bone tissue) in the section by proper tool (such as ‘magic pen’ in the Adobe Photoshop), read out the size (pixels) of the whole red (pink) area. Then pick the whole section area, read out the size (pixels) of the whole implant section area. Calculate bone formation ratio in each section as:
      Bone volume (Bv) = Red area size/Whole section size


      Figure 3. Scanned section

    3. Do the same analysis for all the staining sections for each implant.
    4. Put all the data together in the excel sheet, calculate the mean, standard variation, and perform proper statistic analysis to compare the difference between different groups.

Notes

  1. Mouse MSC can always reach the bone formation ratio 15-30%; while human MSC always has 5-12% bone formation ratio.

Recipes

  1. Formic acid solution
    0.4 M formic acid
    0.5 M sodium formate

References

  1. Abdallah, B. M., Ditzel, N. and Kassem, M. (2008). Assessment of bone formation capacity using in vivo transplantation assays: procedure and tissue analysis. Osteoporosis, Springer: 89-100.
  2. Chen, L., Holmstrom, K., Qiu, W., Ditzel, N., Shi, K., Hokland, L. and Kassem, M. (2014). MicroRNA-34a inhibits osteoblast differentiation and in vivo bone formation of human stromal stem cells. Stem Cells 32(4): 902-912.

材料和试剂

  1. 人或小鼠间充质干细胞(培养,处理或转染)
    注意:如果使用原代骨髓干细胞(来自骨髓培养的附着细胞),最好在低通道(不超过通道3)中使用细胞; 如果使用MSC细胞系,也最好使用低通道而不是高通道。
  2. 小鼠[约8周龄的NOD.CB17-Prkdc scid/J小鼠(NOD/SCID)]
  3. 羟基磷灰石/磷酸三钙(HA/TCP)(Zimmer,目录号:60-130)
  4. 4%多聚甲醛
  5. 苏木精/伊红(H& E)染色溶液
  6. 甲酸溶液(参见配方)

设备

  1. 1 ml注射器
  2. 棉棒(Pro-Ophtha)
  3. 解释器
  4. 机架(用于在细胞培养箱中垂直固定注射器)
  5. 小鼠手术工具(解剖刀,镊子,持针器,剪刀,缝线,无菌拭子,无菌帷帘)
  6. 扫描显微镜(Leica Microsystems或任何扫描显微镜)
  7. 计算机(使用图像分析软件,如ImageJ ®或Photoshop CS ®

软件

  1. ImageJ ®或Photoshop CS ®

程序

  1. 准备植入物(具有HA/TCP支架的基质干细胞)
    1. 取1毫升注射器,用手术刀切断尖端,让注射器尖端侧面打开,但保持活塞在内。
    2. 重量40 mg羟基磷灰石(HA/TCP)并转移到每个注射器。
    3. 放一个切割的棉棒,关闭注射器的开口侧。
    4. 用羟基磷灰石(HA/TCP)高压灭菌注射器。
    5. 取灭菌注射器和HA/TCP回细胞培养室。 将注射器放在架子上(注射器必须保持垂直)。
    6. 打开 注射器,向每个HA/TCP支架中加入100μl10%FBS MEM培养基。 混合   通过1ml尖端上下移液以彻底润湿HA/TCP
    7. 将MSC细胞(处理的或转染的)胰蛋白酶化并计数
    8. 将在200μl培养基中的5×10 5个细胞转移到注射器中的HA/TCP中
    9. 充分混合,但轻轻地用1ml吸头上下移液
    10. 将具有注射器的架子放入培养箱中,37℃,5%CO 2,过夜(见图1)。


      图1。 带注射器的机架

    11. 第二天,将移植细胞在HA/TCP进入动物房。 准备在小鼠皮下移植细胞

  2. 在小鼠中植入
    1. NOD/SCID小鼠,在灭菌的动物室中饲养,约8周龄。 每 鼠标可以在四个位置(右前,左前,   右后,左后)。
      注意:为了避免变化引起 通过单个小鼠或植入位置,表明在每只小鼠,   与对照组和实验组比较; 在每个位置,随机 植入不同处理的样品。
    2. 麻醉小鼠使用一般方案。 让老鼠躺着放松背部上面(见图2A)。
    3. 在脊柱的一侧,在背部中间刮小鼠的毛皮。 乙醇对剃毛区域的皮肤进行消毒。 让它干了一会儿 (参见图2B)。
    4. 在皮肤上切开1-1.5厘米的间隙(见图2C)。
    5. 推动注射器的活塞与细胞内,以摆脱额外的介质上植入物。
    6. 插入1毫升注射器与细胞在HA/TCP中的切口间隙   皮肤。 去皮肤下1.5厘米向前的位置从 切割间隙到达前部植入位置(参见图2D-E)
    7. 推动注射器的活塞,让植入物(带有细胞的HA/TCP) 在皮肤下方的位置放置植入物(见图2E)
    8. 慢慢地将注射器拖出,将其丢弃(参见图2F)。
    9. 重复同样的步骤使用另一种植入物,这个时候让 注射器插入1.5厘米背部皮肤下的背部位置 切割间隙到达背部植入位置
    10. 在前后植入物完成后,缝合切割皮肤(见图2G)。
    11. 在鼠标背面脊柱的另一侧做同样的操作(见图2H)。


      图2。 植入小鼠

    12. 继续,直到所有的植入物放入小鼠
    13. 保持小鼠温暖,并在观察房间过夜观察。
    14. 然后将小鼠转移到常规喂养室(无菌动物室)。
    15. 等待8周,直到植入物中的骨形成。

  3. 植入物的固定,嵌入和H& E染色
    1. 牺牲老鼠,从每个位置取出每个植入物,直接固定在4%多聚甲醛过夜
    2. 弃去多聚甲醛,在4℃下在甲酸中脱钙植入3天,然后在4℃保存在PBS中。
    3. 使用一般方案将植入物植入石蜡中。
    4. 将每个植入物切割成切片(4-μm),在植入物的不同三个位置处切割至少三个切片
    5. 使用一般方案用H& E Y染色所有切片。

  4. 在显微镜下扫描切片
    1. 将切片染色载玻片放在显微镜房间。
    2. 在显微镜下扫描每个部分(整个部分)。
    3. 将扫描的图片保存在磁盘中。

  5. 通过图像分析软件分析骨形成体积
    1. 在Photoshop或其他图像分析中打开每个部分扫描图片 软件。 骨形成区域染色为红色(粉红色),HA/TCP 支架染色为深褐色,非分化的MSC显示为蓝色 斑点。
    2. 选择部分的所有红色区域(骨组织) 正确的工具(如Adobe Photoshop中的"magic pen"),读出 整个红色(粉色)区域的大小(像素)。 然后选择整个部分 区域,读出整个植入部分区域的大小(像素)。 将每个部分的骨形成比计算为:
      骨量(Bv)=红色区域大小/整个区域大小


      图3。 扫描部分

    3. 对每个植入物的所有染色切片进行相同的分析。
    4. 将所有的数据放在excel表中,计算平均值, 标准差,并进行适当的统计分析来比较   不同组之间的差异。

笔记

  1. 小鼠MSC总能达到15-30%的骨形成率; 而人MSC总是具有5-12%的骨形成比。

食谱

  1. 甲酸溶液
    0.4 M甲酸
    0.5M甲酸钠

参考文献

  1. Abdallah,B.M.,Ditzel,N。和Kassem,M。(2008)。 使用在体内评估骨形成能力 移植试验:程序和组织分析。骨质疏松,Springer:89-100。
  2. Chen,L.,Holmstrom,K.,Qiu,W.,Ditzel,N.,Shi,K.,Hokland,L.and Kassem,M.(2014)。 MicroRNA-34a抑制成骨细胞分化和体内人类基质的骨形成 干细胞。 干细胞 32(4):902-912。
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How to cite this protocol: Chen, L. and Ditzel, N. (2015). In vivo Heterotopic Bone Formation Assay Using Isolated Mouse and Human Mesenchymal Stem Cells. Bio-protocol 5(3): e1389. DOI: 10.21769/BioProtoc.1389; Full Text



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