Experimental study of subcutaneous adipose-derived stem cells inhibiting orthodontic root resorption
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摘要:目的 探讨人皮下脂肪来源干细胞（human subcutaneous adipose-derived stem cells，hADSCs）局部移植对正畸源性牙根吸收（orthodontically induced root resorption，OIRR）的影响，为临床应用hADSCs抑制OIRR提供实验依据。方法 取40只8周龄雄性SD大鼠随机分为实验组和对照组，每组20只，建立大鼠右侧上颌第1磨牙近中牙正畸牙移动（orthodontic tooth movement，OTM）模型。实验组大鼠于建模第1、4、8、12天注射25 μL含2.5×105个hADSCs的细胞悬液，对照组注射25 μL PBS。在加力前及加力7、14 d后获取大鼠上颌模型，于加力7、14 d后两组各处死10只大鼠并取材。体式显微镜测量OTM距离，扫描电镜观察压力侧牙根形态及测定牙根吸收面积比，HE染色观察压力侧牙根吸收及牙周组织改建并计算牙根吸收指数，抗酒石酸酸性磷酸酶染色计数压力侧牙周组织破牙骨质细胞和破骨细胞数量。结果 两组OTM距离均随加力时间延长而增加（P<0.05）；加力7、14 d实验组和对照组比较OTM距离差异无统计学意义（P>0.05）。扫描电镜观察示加力7 d，实验组和对照组牙根表面见小而浅的散在吸收陷窝，两组牙根吸收面积比差异无统计学意义（P>0.05）；加力14 d，两组牙根吸收陷窝加深变大，实验组牙根吸收面积比显著小于对照组（P<0.05）。实验组牙根吸收范围小于对照组、深度浅于对照组，加力14 d实验组牙根吸收指数显著小于对照组（P<0.05）。加力7、14 d实验组破牙骨质细胞计数均显著少于对照组（P<0.05）；加力14 d实验组破骨细胞计数显著少于对照组（P<0.05）。结论 hADSCs 局部移植可能通过降低大鼠OTM过程中破牙骨质细胞和破骨细胞的数量来减小牙根吸收面积和深度，从而抑制OIRR。
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关键词:
- 正畸源性牙根吸收 /
- 人皮下脂肪来源干细胞 /
- 正畸牙移动 /
- 破牙骨质细胞 /
- 破骨细胞
Abstract:Objective To investigate the effect of human subcutaneous adipose-derived stem cells (hADSCs) local transplantation on orthodontically induced root resorption (OIRR) and provide theoretical and experimental basis for the clinical application of hADSCs to inhibit OIRR.Methods Forty 8-week-old male Sprague Dawley rats were randomly divided into experimental group and control group, with 20 rats in each group, to establish the first molar mesial orthodontic tooth movement (OTM) model of rat right maxillary. The rats in the experimental group were injected with 25 μL of cell suspension containing 2.5×105 hADSCs on the 1st, 4th, 8th, and 12th day of modeling, while the rats in the control group were injected with 25 μL of PBS. The rat maxillary models were obtained before and after 7 and 14 days of force application, and 10 rats in each group were killed and sampled after 7 and 14 days of force application. The OTM distance was measured by stereomicroscope, the root morphology of the pressure side was observed by scanning electron microscope and the root resorption area ratio was measured. The root resorption and periodontal tissue remodeling of the pressure side were observed by HE staining and the root resorption index was calculated. The number of cementoclast and osteoclast in the periodontal tissue on the pressure side was counted by tartrate resistant acid phosphatase staining.Results The TOM distance of both groups increased with the extension of the force application time, and there was no significant difference (P<0.05). There was no significant difference in OTM distance between the experimental group and the control group after 7 and 14 days of force application (P>0.05). Scanning electron microscope observation showed that small and shallow scattered resorption lacunae were observed on the root surface of the experimental group and the control group after 7 days of force application, and there was no significant difference in the root resorption area ratio between the two groups (P>0.05); after 14 days of application, the root resorption lacunae deepened and became larger in both groups, and the root resorption area ratio in the experimental group was significantly lower than that in the control group (P<0.05). The range and depth of root absorption in the experimental group were smaller and shallower than those in the control group, and the root absorption index in the experimental group was significantly lower than that in the control group after 14 days of force application (P<0.05). The number of cementoclast in the experimental group was significantly lower than that in the control group after 7 and 14 days of force application (P<0.05); the number of osteoclasts in the experimental group was significantly lower than that in the control group after 14 days of force application (P<0.05).Conclusion Local transplantation of hADSCs may reduce the area and depth of root resorption by reducing the number of cementoclasts and osteoclasts during OTM in rats, thereby inhibiting orthodontic-derived root resorption. -
图 1 hADSCs 的分离、培养及鉴定
a. 细胞复苏培养3 d（倒置相差显微镜×40）；b～g. 流式细胞检测（分别为CD45、CD90、CD105、CD29、CD73、CD44）；h. 成骨诱导培养3周茜素红染色（倒置相差显微镜×200）；i. 成脂诱导培养3周油红O染色（倒置相差显微镜×200）；j. 成软骨诱导培养4周阿利新蓝染色（正置显微镜×200）
Figure 1. Isolation, culture, and identification of hADSCs
a. Cell resuscitation and culture for 3 days (Inverted phase contrast microscope×40); b-g. Flow cytometry detection (CD45, CD90, CD105, CD29, CD73, CD44, respectively); h. Alizarin red staining at 3 weeks after osteogenic induction (Inverted phase contrast microscope×200); i. Oil red O staining at 3 weeks after adipogenic induction (Inverted phase contrast microscope×200); j. Alcian blue staining at 4 weeks after chondrogenic induction (Upright microscope×200)
图 2 上颌第1磨牙远中根近中面扫描电镜观察（×45）
a. 左侧未加力侧；b. 对照组（左）和实验组（右）加力7 d（箭头示吸收陷窝）；c. 对照组（左）和实验组（右）加力14 d（箭头示吸收陷窝）
Figure 2. Scanning electron microscope observation of the mesial surface of the distal root of the maxillary first molar (×45)
a. The left side without force application; b. The control group (left) and the experimental group (right) applied force for 7 days (arrow showed the absorption lacuna); c. The control group (left) and the experimental group (right) applied force for 14 days (arrow showed the absorption lacuna)
图 3 HE染色观察（×200）
AB：牙槽骨 PDL：牙周膜TR：牙根 a. 左侧上颌第1磨牙牙周组织；b. 对照组（左）和实验组（右）加力7 d（上）和14 d（下）压力侧牙根和牙周组织；c. 对照组（左）和实验组（右）加力7 d（上）和14 d（下）张力侧牙根和牙周组织
Figure 3. HE staining observation (×200)
AB: Alveolar bone PDL: Periodontal ligament TR: Tooth root a. Periodontal tissue of the left maxillary first molar; b. Root and periodontal tissue of the pressure side of the control group (left) and the experimental group (right) after 7 days (upper) and 14 days (lower) of force application; c. Root and periodontal tissue of the tension side of the control group (left) and the experimental group (right) for 7 days (upper) and 14 days (lower) of force application
图 4 两组压力侧牙根吸收指数计算示意图（HE×100）
TR：牙根 黑色示牙根吸收区域，灰色示牙根未吸收区域 a. 对照组加力7 d；b. 实验组加力7 d；c. 对照组加力14 d；d. 实验组加力14 d
Figure 4. Schematic diagram of calculation of root resorption index on the pressure side of the two groups (HE×100)
TR: Tooth root Black area indicated the root resorption area, and gray area indicated the root non-resorption area a. The control group applied force for 7 days; b. The experimental group applied force for 7 days; c. The control group applied force for 14 days; d. The experimental group applied force for 14 days
图 5 上颌第1磨牙牙根近中侧TRAP染色观察
AB：牙槽骨 PDL：牙周膜 TR：牙根 a. 左侧未加力侧（×400）；b. 对照组（左）和实验组（右）加力7 d（上）和14 d（下）（×200） 蓝箭头示破牙骨质细胞，绿箭头示破骨细胞
Figure 5. TRAP staining observation of the mesial side of the root of the maxillary first molar
AB: Alveolar bone PDL: Periodontal ligament TR: Tooth root a. Left unforced side (×400); b. The control group (left) and the experimental group (right) applied force for 7 days (upper) and 14 days (lower) (×200) Blue arrow showed the cementoclasts, green arrow showed the osteoclasts
表 1 加力7、14 d实验组和对照组各指标比较（n=10，
${ \overline {\boldsymbol{x}} \pm {\boldsymbol{s}}} $ ）Table 1. Comparison of indicators between the experimental group and the control group after 7 and 14 days of force application (n=10,
${ \overline {\boldsymbol{x}} \pm {\boldsymbol{s}}} $ )组别
GroupOTM距离（mm）
OTM distance (mm)牙根吸收面积比
Root resorption area ratio牙根吸收指数
Root resorption index7 d 14 d 统计值
Statistic7 d 14 d 统计值
Statistic7 d 14 d 统计值
Statistic对照组
Control group0.050±0.035 0.121±0.045 t=–3.957
P=0.0010.021±0.009 0.198±0.055 t=–7.130
P<0.0010.009±0.012 0.050±0.006 t=–6.879
P<0.001实验组
Experimental group0.035±0.024 0.112±0.038 t=–5.380
P<0.0010.016±0.015 0.111±0.032 t=–6.001
P<0.0010.008±0.005 0.034±0.009 t=–5.832
P<0.001统计值
Statistict=–1.156
P=0.263t=–0.533
P=0.601t=–0.634
P=0.544t=–3.058
P=0.016t=–0.233
P=0.829t=–3.453
P=0.009
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组别
Group破牙骨质细胞数（个/视野）
Cementoclasts (cells/field)破骨细胞数（个/视野）
Osteoclasts (cells/field)7 d 14 d 统计值
Statistic7 d 14 d 统计值
Statistic对照组
Control group4.188±1.693 3.200±1.479 t=0.982
P=0.3555.688±1.493 6.300±1.674 t=–0.611
P=0.558实验组
Experimental group1.950±0.524 0.800±0.695 t=2.955
P=0.0184.625±1.243 3.688±1.353 t=1.141
P=0.287统计值
Statistict=–2.824
P=0.022t=–3.285
P=0.011t=–1.223
P=0.256t=–2.714
P=0.027
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