Value of allogeneic adipose stem cells combined with demineralized bone matrix in rabbit model of critical bone defect of radius
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摘要:目的 探讨同种异体脂肪干细胞复合脱钙骨基质对桡骨临界骨缺损兔模型的价值。方法 纳入18只成年雄性大白兔，切取双上肢桡骨干中段部分骨质，制备双侧上肢桡骨临界骨缺损模型。将18只兔子随机分为对照组、实验组1和实验组2。对照组未予任何干预，实验组1在骨缺损部位植入脱钙骨基质，实验组2在骨缺损部位植入同种异体脂肪干细胞复合脱钙骨基质。第8、12周时行X线、病理切片和解剖学检查，明确骨缺损的修复效果。结果 骨缺损修复后, 8、12周时对桡骨行X线检查，对照组骨缺损修复差; 8周时实验组1部分修复, 12周时大部分修复，但仍有1只兔子存在小块骨缺损; 8周时实验组2大部分修复, 12周时全部修复，实验组2可见髓腔通畅。8、12周时，实验组1和实验组2的Lane-Sandhu组织学评分均显著高于对照组，实验组2的Lane-Sandhu组织学评分均显著高于实验组1(P < 0.05)。造模12周后，对照组骨缺损区多为纤维组织，实验组1有编织骨形成，但骨小梁紊乱; 实验组2有板成骨形成，骨小梁排列紧密有序。结论 同种异体脂肪干细胞复合脱钙骨基质对桡骨临界骨缺损兔模型有一定的价值。Abstract:Objective To investigate the value of allogeneic adipose stem cells combined with demineralized bone matrix in rabbit model of critical bone defect of radius.Methods A total of 18 adult male rabbits were collected and treated with dissection of partial bone in the middle segment of the radial diaphysis of the upper limb, and the rabbit model of critical bone defect of radius of the bilateral upper limb was prepared. The 18 rabbits were randomly divided into control group, experimental group 1 and experimental group 2. The control group was not given any intervention, and the experimental group 1 was implanted with the demineralized bone matrix in the bone defect site, while the experimental group 2 was implanted with the allograft adipose stem cells and the decalcified bone matrix. The X-ray, pathological section and anatomical examination were performed at 8 and 12 weeks to clarify the repair effect of bone defect.Results After repairing the bone defect, X-ray examination was performed on the radius at 8 and 12 weeks. The bone defect in the control group was poorly repaired. At 8 weeks, part of the bone defect was repaired in the experimental group 1, and most of the bone defect was repaired at 12 weeks, but there was still a small bone defect in a rabbit. At 8 weeks, most of the bone defect was repaired in the experimental group 2, and all of them were repaired at 12 weeks. At 8 and 12 weeks, the Lane-Sandhu histological scores of the experimental group 1 and group 2 were significantly higher than that of the control group, and the Lane-Sandhu histological score of the experimental group 2 was significantly higher than that of the experimental group 1 (P < 0.05). After 12 weeks, the bone defect area in the control group was mostly fibrous tissue, while in the experimental group 1, woven bone was formed, but bone trabecula was disordered. In the experimental group 2, plate osteogenesis was formed, and bone trabeculae were arranged tightly and orderly.Conclusion Allogeneic adipose stem cells combined with demineralized bone matrix have a certain therapeutic value in the rabbit model of critical bone defect of radius.
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Keywords:
- allogeneic adipose stem cells /
- radius /
- bone defect /
- demineralized bone matrix
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骨缺损可继发于创伤、感染等，引起患者关节功能障碍，而骨缺损的修复是临床上的重点和难点问题[1-3]。传统的治疗方法包括骨移植(自体骨移植或异体骨移植)和非骨性材料移植等，但存在并发症多、效果不理想等问题。自体骨移植效果尚可，但增加了患者创伤，而异体骨移植供骨有限，且存在免疫排斥反应。脂肪干细胞在一定条件下可分化为成骨细胞，其成骨能力较强，可以作为种子细胞的重要来源[4-7]。
1. 资料与方法
1.1 实验动物和分组
纳入18只健康的成年雄性新西兰大白兔，切取双上肢桡骨干中段部分骨质，制备双侧上肢桡骨临界骨缺损模型。将18只兔子随机分为对照组、实验组1和实验组2。3组体质量依次为(3.26±0.23)、(3.22±0.21)和(3.28±0.26) kg, 差异无统计学意义(P>0.05)。实验动物均由安徽医科大学实验动物中心提供。
1.2 模型制作
3%戊巴比妥经耳缘静脉注射(1 mL/kg), 麻醉后固定于手术台，无菌条件下双上肢做约3 cm的纵形切口，分离肌间隙，充分暴露桡骨，剥离骨膜，锯条切取长25 mm的桡骨形成桡骨缺损模型。术毕由同一饲养员分笼饲养。
1.3 脂肪干细胞提取
取实验兔腹股沟皮下脂肪组织，充分剪碎后加入0.075%Ⅰ型胶原酶37℃水浴箱震动30 min, 2 600转/min离心去除上清和漂浮脂肪，加入DMEM生长液，接种于25 cm2的培养瓶中，培养箱内培养, 48 h更换培养液1次。细胞覆盖90%瓶底时，胰蛋白酶消化、传代，取第3代脂肪干细胞，贴壁完全后在原DMEM的基础上，将维生素C、β-甘油磷酸钠、地塞米松加入，诱导成骨，成骨诱导因子为1 mg/L的骨形态发生蛋白2。见图 1。
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图 1 脂肪干细胞的培养及传代(放大倍数100倍)A、B、C、D依次表示原代脂肪干细胞第1、3、5、7天; E、F、G、H依次表示第2代脂肪干细胞第3、5、7、10天; I、J依次代表第3代脂肪干细胞第7、10天1.4 脱钙骨基质制作
取兔髂骨，剔除软组织、骨膜和一侧骨皮质(一侧为松质骨，对侧为皮质骨的骨块)，冲洗骨髓和血迹，多次蒸馏水漂洗，晾干, 1:1氯仿甲醇溶液室温下浸泡脱脂2次(每6 h 1次)，风干, 0.6 mol/L HCl、4 ℃脱钙, 6 h更换液体1次(50 mL/g), 风干骨块，检查脱钙情况(不坚硬且有弹性)，蒸馏水冲洗至中性, 1:1氯仿甲醇溶液脱脂1次, 37 ℃恒温箱下干燥后-20 ℃冰箱保存。使用前将其剪成4 mm×4 mm×2 mm的小方块，体积分数75%的乙醇浸泡30 min, 过夜晾干后紫外线照射30 min, DMEM预湿24 h。
1.5 脂肪干细胞与脱钙骨基质复合培养
制成脂肪干细胞浓度为4×108个/L的悬液，取预湿支架，无菌纱布吸干, 30 μL均匀接种于脱钙骨基质的松质鼓面，培养箱中培养，支架周围加入培养基, 2 d更换1次培养基, 1周后观察细胞黏附和生长情况。
1.6 DIO荧光标记观测细胞在材料表面的增殖以及细胞在三维材料上的增殖曲线
将第3代脂肪干细胞(rADSCs)预先用3, 3-Dioctadecylox-acarbocyanine-perchlorate(DIO)荧光染料标记，接种于材料上并在诱导培养液中共培养。将无血清LG-DMEM培养液以1:250的比例稀释DIO染液(1 mmol/L), 将稀释后的染液重悬第3代rADSCs, 调整细胞浓度为1×106 cells/mL, 混匀后在CO2培养箱中孵育20 min, 1 000转/min离心10 min, 去上清液，以新鲜生长培养液重悬细胞；调整细胞密度为3×105 cells/mL, 接种于脱钙骨材料表面(同一批次)，于细胞培养箱中培养4 h后，加入生长培养液；第2天更换成骨诱导液培养，分别于接种3、7 d后采用荧光显微镜与共聚焦显微镜观察细胞在三维支架上的生长情况，见图 2。
将第3代rADSCs以3×105 cells/mL密度接种于脱钙骨材料上(同一批次)，培养箱孵育4 h后加入GM, 第2天更换为成骨诱导液。以生长培养液作为对照组。分别于接种1、3、5、7、9、12、15 d后收集材料上的细胞，以Hoechst 33258 DNA定量方法对rADSCs进行细胞计数，对未接种细胞的脱钙骨材料进行相同的处理，所测值作为空白对照，并从各检测值中减去。利用测得荧光强度的吸光度(OD)值来反映细胞的生长趋势，即OD值越大代表细胞生长趋势越好。见图 3。
1.7 干预方式
对照组未予任何干预，实验组1在骨缺损部位植入脱钙骨基质，实验组2在骨缺损部位植入同种异体脂肪干细胞复合脱钙骨基质。第8、12周时行X线、病理切片和解剖学检查，明确骨缺损的修复效果。
1.8 观察指标
Lane-Sandhu组织学评分标准评分、放射学评价、解剖学形态和兔桡骨标本苏木精-伊红染色光镜下结构。
1.9 统计学分析
采用SPSS 22.0完成数据分析, 3组计量资料比较采用方差分析，两两比较采用SNK检验。
2. 结果
2.1 实验动物不良反应和生存情况
对照组1只兔切口部位裂开，缝合后愈合良好，实验期间无动物意外死亡。18只兔子全部纳入结果分析。
2.2 放射学评价
骨缺损修复后, 8、12周时对桡骨行X线检查，对照组骨缺损修复差; 8周时实验组1部分修复, 12周时大部分修复，但仍有1只兔子存在小块骨缺损; 8周时实验组2大部分修复, 12周时全部修复，实验组2可见髓腔通畅。见图 4。
2.3 Lane-Sandhu组织学评分评价
8、12周时，实验组1和实验组2的Lane-Sandhu组织学评分均高于对照组，实验组2的Lane-Sandhu组织学评分均高于实验组1, 差异均有统计学意义(P < 0.05)。见表 1。
表 1 Lane-Sandhu组织学评分评价(x±s)分 组别 n 8周时 12周时 对照组 6 1.86±0.65 2.32±0.81 实验组1 6 4.73±1.12* 8.47±1.62* 实验组2 6 6.12±1.53*# 11.47±1.47*# 与对照组比较, *P < 0.05;与实验组1比较, P < 0.05。 2.4 兔桡骨标本苏木精-伊红染色评价
造模12周后对照组骨缺损区多为纤维组织，实验组1有编织骨形成，但骨小梁紊乱; 实验组2有板成骨形成，骨小梁排列紧密有序。见图 5。
2.5 桡骨缺损的解剖学观察
12周时对照组、实验组1均有不同程度的缺损，实验组2骨形态良好。见图 6。
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图 6 桡骨缺损的解剖学观察A、B、C分别为对照组0、8、12周桡骨缺损的形态; D、E、F分别为实验组1在0、8、12周桡骨缺损的形态; G、H、I分别为实验组2在0、8、12周桡骨缺损的形态3. 讨论
目前骨缺损修复的手术已经较为成熟，骨的来源成为制约其进一步发展的瓶颈，主要原因是自体骨移植创伤较大，取材受限，尤其是大块骨缺损，异体骨移植也存在供骨有限的问题，且可能会导致排斥反应。组织工程学的快速发展为突破这一瓶颈提供了基础。本研究探讨同种异体脂肪干细胞联合脱钙骨基质对桡骨临界骨缺损兔模型的价值，结果发现同种异体脂肪干细胞联合脱钙骨基质对桡骨临界骨缺损兔模型具有较好的修复价值，效果比单纯使用脱钙骨基质好。
干细胞是可以自我复制且具备多向分化潜能的原始细胞，是机体形成各种组织中原始细胞。干细胞技术又称再生医学，是医学生物治疗中最前沿的技术之一。近年来，脂肪干细胞被发现具有多种分化潜能，如在骨形态发生蛋白的诱导下，脂肪干细胞可以分化为成骨细胞[8-12]。本研究也证实从兔子脂肪细胞中提取的细胞生长能力旺盛，可以分化为成骨细胞，具有干细胞的特性。对于骨缺损患者而言，通过自身的脂肪干细胞构建具有完整生物学结构和功能正常脂肪组织无疑是解决供骨的来源不可回避的问题。脂肪干细胞自被发现以来，就被证实具有向软骨、成骨、成肌等多向分化潜能[6, 13-18]。本研究通过在兔桡骨骨折部位移植具有向成骨细胞分化倾向的脂肪干细胞，可以促进成骨细胞在骨折部位的大量形成，促进骨折愈合。目前已有学者[19-22]验证脂肪干细胞用于骨缺损模型的修复，具有促进骨折愈合的过程，支持本研究结果。
本研究将脂肪干细胞和脱钙骨基质复合，脱钙骨基质是天然骨移植材料，包括胶原蛋白、非胶原蛋白、生长因子，具有诱导成骨的能力，可以促进新骨形成及骨组织矿化，加速骨愈合，可以单独或与自体骨、其他生物材料、生长因子联合有效修复骨损伤，是比较理想的骨组织工程支架材料[23-24], 可以来源于人或动物的股骨干和胫骨干等。本研究将脂肪干细胞和脱钙骨基质复合，可促进成骨细胞的大量繁殖，在脱钙骨基质的作用下，成骨细胞的成骨能力可以得到进一步提升，极大地促进了新骨形成及骨组织矿化，加速骨愈合，效果较单纯应用脱钙骨基质好。
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图 1 脂肪干细胞的培养及传代(放大倍数100倍)
A、B、C、D依次表示原代脂肪干细胞第1、3、5、7天; E、F、G、H依次表示第2代脂肪干细胞第3、5、7、10天; I、J依次代表第3代脂肪干细胞第7、10天
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图 6 桡骨缺损的解剖学观察
A、B、C分别为对照组0、8、12周桡骨缺损的形态; D、E、F分别为实验组1在0、8、12周桡骨缺损的形态; G、H、I分别为实验组2在0、8、12周桡骨缺损的形态
表 1 Lane-Sandhu组织学评分评价(x±s)
分 组别 n 8周时 12周时 对照组 6 1.86±0.65 2.32±0.81 实验组1 6 4.73±1.12* 8.47±1.62* 实验组2 6 6.12±1.53*# 11.47±1.47*# 与对照组比较, *P < 0.05;与实验组1比较, P < 0.05。 
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