Repairing articular cartilage defects in rabbits by adipose-derived stem cells combined with chitosan/collagen scaffold and GDF-5

doi:10.3969/j.issn.2095-4344.0955

Abstract

Abstract:

BACKGROUND: GDF-5 can induce the differentiation of human adipose-derived stem cells into chondrocyte-like cells, and chitosan/collagen scaffolds are widely used in cartilage tissue repair.
OBJECTIVE: To repair rabbit articular cartilage defects by using rabbit autologous adipose-derived stem cells combined with chitosan/collagen scaffold and GDF-5 and to observe the repair effect.
METHODS: Adipose-derived stem cells isolated from subcutaneous adipose tissues of Japanese rabbits were cultured, purified, amplified in vitro, and labeled with BrdU in vitro. The chitosan/collagen composite scaffold was prepared in a ratio of 1:3 using ultraviolet cross-linking method. Then labeled adipose-derived stem cells were seeded onto the scaffold and cultured to form complexes. A cylindrical defect (5 mm in diameter, 3 mm in depth) in the knee articular cartilage was surgically made in each rabbit, and these rabbit models were thereafter randomly divided into four groups. Composite group: adipose-derived stem cells+chitosan/collagen scaffold+GDF-5 complex implantation; stent group: simple implantation of chitosan/collagen scaffold; cell group: simple implantation of adipose-derived stem cells; and sham operation group: simple drilling without treatment. All the rabbits were intervened for 4, 8, and 12 weeks. Meanwhile, histological detection, type II collagen immunohistochemical staining and BrdU mmunofluorescence staining were performed.
RESULTS AND CONCLUSION: (1) At 12 weeks after surgery, the cylindrical defect of the composite group was completely filled with the same milky white transparent substance as the surrounding normal cartilage, and the cartilage defect area was replaced by differentiated hyaline cartilage. Of all the groups, Wakitani score was the lowest in the composite group (P < 0.01). (2) Type II collagen immunohistochemical staining and BrdU immunofluorescence staining: in the composite group, the type II collagen stained cytoplasm in the repaired area was detected by immunohistochemical staining at 4, 8, and 12 weeks after surgery. The expression of BrdU in the new tissues of the repaired area was positive. These results confirm that autologous adipose-derived stem cells+chitosan/collagen scaffold+GDF-5 complex grafting has a good effect on the repair of rabbit knee cartilage defects.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Tissue Engineering, Chitosan, Stents

CLC Number:

R394.2

Wang Zheng, Yu Hui, Yu Yao, Wu Bo-lin. Repairing articular cartilage defects in rabbits by adipose-derived stem cells combined with chitosan/collagen scaffold and GDF-5[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 3969-3974.

Figures/Tables 1

2.1 实验动物数量分析共纳入日本大耳兔72只，死亡3只，补充3只，最终进入结果分析的兔72只。 2.2 壳聚糖/胶原蛋白生物支架理化性质及体内相容性壳聚糖/胶原蛋白支架孔隙丰富，大小比较均一，且孔与孔之间相互联通构成了交通孔。支架的孔径在90-360 μm (图1A)；各组孔径经均数比较均有差异(P < 0.05)；各组支架孔隙率、吸水率等各项理化指标良好(表1)。实验将支架植入大耳兔腹部组织皮下经苏木精-伊红染色可见支架生物组织相容性较好(图1B，C)。 2.3 术后兔一般情况观察及修复后组织学评分兔术后第1天切口处略红肿，关节略肿胀，术后3-7 d切口处无红肿及关节肿胀明显消退。复合物组术后4，8，12周Wakitani评分复合物组评分最低(P < 0.01)；支架组与脂肪干细胞组，假手术组比较差异有显著性意义(P < 0.01)，见表2。 2.4 关节软骨修复组织学及形态学变化移植物软骨缺损修复术后，随时间延长，复合物组患肢恢复从组织学检查结果和一般观察情况明显好于支架组及脂肪干细胞组，至术后12周，复合物组：大体观察圆柱型缺损处完全被与周围正常软骨相同的乳白色透明样物质填充修复(图2A)。组织学染色显示：软骨缺损区域由分化一致的透明软骨所替代修复(图1D)；支架组缺损处由纤维软骨所替代(图1E，2B)；脂肪干细胞组及假手术组由少量纤维组织部分填充(图1F，2C)Ⅱ型胶原免疫组织化学染色证实：复合物组与支架组4，8，12周免疫组织化学染色可见修复区组织Ⅱ型胶原染色胞浆内呈淡黄色阳性表达；脂肪干细胞组及假手术组表达呈阴性；复合物组术后4，8，12周修复区新生组织进行BrdU免疫荧光染色，可见绿色荧光表达(图3)。支架组与脂肪干细胞组及假手术组未见绿色荧光表达。"

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