人羊膜间充质干细胞片封装软骨颗粒促进兔骨软骨缺损的修复

关节骨软骨缺损是疼痛和残疾的重要原因。骨软骨缺损不仅局限于局部创伤，还可能导致整个关节疾病。已有研究表明，骨软骨缺损或全层软骨缺损可引起关节软骨丢失和早期骨关节炎发生。由于软骨的无血管特性，成年软骨损伤后愈合能力非常有限。目前已经尝试了几种手术治疗方法来修复关节表面缺陷，包括自体软骨植入、骨软骨移植(自体或异体)和无细胞仿生支架植入等，但对于最有效的治疗方式还没有定论。

近年来，有研究报道了青少年新鲜幼软骨颗粒的应用。该技术消除了与自体移植相关的供体部位的发病率。目前，这项技术已被用于修复膝关节、髋关节和踝关节软骨缺损。该技术短期效果明显，但仍存在一些挑战，包括软骨下水肿和微粒软骨所放位置的软骨表面不均匀等。此外，细胞片层技术也被用于关节软骨缺损的治疗。与其他细胞片相比，软骨细胞片在软骨缺损修复中的应用研究较多，但软骨细胞在体外培养时很容易分化。当去分化软骨细胞被移植到软骨缺损时，再生组织最终会变成纤维软骨，与正常软骨相比，纤维软骨的功能较差。人羊膜间充质干细胞(hAMSCs)以其高活性、高纯度、高增殖、低免疫原性而受到研究者的关注。hAMSCs在实验室条件下具有很高的软骨分化潜能，在软骨再生方面具有巨大的应用潜力。

据此，遵义医学院的刘毅教授团队探讨了包裹软骨颗粒的hAMSCs细胞片是否有助于兔骨软骨缺损的修复（图1）。研究假设，与仅使用软骨颗粒处理的缺陷相比，以细胞片形式联合使用软骨颗粒能更快的促进骨软骨修复和软骨再生。

Figure 1. Schematic diagram of the experimental operation. hAMSCs, human amniotic mesenchymal stem cells.

研究者首先对hAMSCs进行了培养和表征。流式细胞术检测发现，第3代hAMSCs高表达间充质标志物CD44、CD73、CD90、CD105，而造血标志物CD19、CD34、CD45、CD11b、HLA-DR几乎不表达(图2B)。免疫荧光显示间充质干细胞标记物vimentin高表达，而人羊膜上皮细胞特异性表型标记物细胞角蛋白19（CK-19）不表达(图2C)。

Figure 2. (A) Microscopic observations of passage 0-2 hAMSCs. Scale bar: 200 μm. (B) hAMSCs were positive for CD44, CD73, CD90, and CD105 and negative for CD19,CD34, CD45, CD11b, and HLA-DR. (C) Passage 3 hAMSCs hardly expressed CK-19. hAMSCs were positive for vimentin. Scale bar: 100 μm. hAMSCs, human amniotic mesenchymal stem cells.

hAMSCs在细胞片诱导培养基中培养14天后形成细胞片，研究者对其结构、特性和形态进行了表征。倒置相差显微镜下，hAMSCs呈长梭形，排列紧密，分布均匀(图3A)。扫描电镜(SEM)结果显示，细胞片呈多层结构，细胞嵌入自身分泌的细胞外基质中(图3B)。流式细胞术结果显示，间充质标记物CD44、CD73、CD90、CD105在细胞片培养基中诱导2周后仍高表达，而CD34、CD11B、CD19、CD45和HLA-DR几乎不表达(图3C)。

Figure 3. (A) Gross and morphological observations of an hAMSC sheet. Scale bars: (b) 100 μm and (c) 50 μm. (B: a, b) Cell sheets had a multilayered structure. (c) Cells were embedded in a large amount of extracellular matrix secreted by themselves. Scale bars: (a) 3μm, (b) 2 μm, and (c) 1 μm. (C) Mesenchymal markers CD44, CD73, CD90, and CD105 were still highly expressed in the hAMSC sheet, and CD34, CD11B, CD19, CD45, and HLA-DR were hardly expressed. hAMSC, human amniotic mesenchymal stem cell.

在软骨诱导培养基中诱导14天后，培养板底部形成白色膜质，圆形或椭圆形细胞排列紧密，分布均匀，镜下可见铺路石结构(图4A)。苏木精、伊红染色显示大量圆形细胞排列紧密，分布均匀。甲苯胺蓝染色显示细胞外基质分泌大量聚集蛋白聚糖。免疫组化染色可见细胞外基质分泌大量II型胶原(图4B)。与非诱导的细胞片组相比，软骨诱导组II型胶原(6.07倍)、SOX9(3.85倍)和Aggrecan(3.96倍)基因表达显著增加(图5A), 蛋白表达水平也显著增加(图5,B和C)。

Figure 4. (A) Macroscopic and morphological observations of chondrogenically induced cell sheets. Scale bars: (b) 200 μm and (c) 100 μm. (B) Cell staining of chondrogenically induced cell sheets. Scale bar: 20 μm. (C) Histological staining of chondrogenically induced cell sheets. Scale bar: 50 μm. HE, hematoxylin and eosin.

Figure 5. mRNA and protein levels of Sex determining region Y-box9 (SOX9), aggrecan (ACAN), and collagen type II (COLII). (A) The mRNA expression of COL II, ACAN, and SOX9 was increased in the induced group on day 14 as compared with the non-induced group. *P <0.05. (B) Relative protein expression levels with and without induction on day 14 as determined by Western blotting. The relative expression of proteins in the induced group was higher than that in the noninduced group. *P<0.05. Data are presented as mean ± SD.

研究者设置了hAMSC细胞片/软骨颗粒、软骨颗粒、hAMSC细胞片和纤维蛋白胶四组用于体内骨软骨缺损的修复研究。结果发现，与其他组相比，hAMSC细胞片/软骨颗粒组修复组织肉眼评分较高；在hAMSC细胞片组中，新生不透明的白色再生组织表现出中等程度的充盈，边界可以被识别；而纤维蛋白胶组缺损区仅见少量新生组织，边缘清晰(图6A)。

3个月时，hAMSC细胞片/软骨颗粒组的缺损区域几乎完全被再生组织填满，safranin O和II型胶原染色呈阳性，表明软骨细胞外基质的存在。再生组织与邻近正常软骨融合良好，可见软骨下骨。hAMSC细胞片组修复组织较hAMSC细胞片/软骨颗粒组更薄。软骨颗粒组软骨修复明显，但再生组织中存在少量空洞，软骨下骨再生效果不佳。纤维蛋白胶组修复组织的组织学染色较弱且在缺陷边缘有明显的轻微退化(图7A)。3个月时，在hAMSC细胞片/软骨颗粒和hAMSC细胞片组骨软骨缺损区观察到大量表达人核特异性抗原的细胞，部分细胞表达SOX9(图7C)。

Figure 6. Macroscopic observation of regenerated tissue. (A) (a) Osteochondral defects (diameter, 3.5 mm; depth, 3 mm) were created in the patellar grooves of experimental animals: (b) hAMSC sheet/cartilage particle group, (c) cartilage particle group, (d) hAMSC sheet group, and (e) fibrin glue group. In the hAMSC sheet/cartilage particle group, the defect area was almost completely filled with a white semitranslucent tissue at 3 months postoperatively, and the boundary connected to the normal cartilage tissue had almost completely disappeared. White circle indicates the boundary of defects. (B) Mean International Cartilage Repair Society macroscopic scores of groups at 3 months after surgery. *P <0.05. **P <0.01. Data are presented as mean ± SD. hAMSC, human amniotic mesenchymal stem cell.

Figure 7. (A) Histological evaluation of regenerated tissue in all groups at 3 months postoperatively: (a) fibrin glue group, (b) hAMSC sheet group, (c) cartilage particle group, (d) hAMSC sheet/cartilage particle group. P, boundary of defects. Scale bar: 200 μm. (B) Mean O Driscoll histological scores of groups at 3 months postoperatively. *P <0.05. **P < 0.01. (C) A largea mount of cells expressing human nuclear-specific antigen (green) were observed in the defect area of group A (hAMSC sheet group) and group B (hAMSC sheet/cartilage particle group), and some cells expressed SOX9 (red). Scale bar: 20 μm. Data are presented as mean ± SD. hAMSC, human amniotic mesenchymal stem cell.

本研究由遵义医学院刘毅教授团队完成，并于2020年1月15日在线发表于The American Journal of Sports Medicine。

 

论文信息: Qi You, Ziming Liu, Jun Zhang, Mengjie Shen, Yuwan Li, Ying Jin, and Yi Liu*. Human Amniotic Mesenchymal Stem Cell Sheets Encapsulating Cartilage Particles Facilitate Repair of Rabbit Osteochondral Defects. The American Journal of Sports Medicine 2020, doi.org/10.1177/0363546519897912.

供稿：余颖康

审校：袁章琴

编辑：丁路光