Correlation between chondrocyte migration and integration capability during autologous chondrocyte transplantation

doi:10.3969/j.issn.2095-4344.2013.44.011

Abstract

Abstract:

BACKGROUND: In joint surgery, the commonly used autologous chondrocyte transplantation often used to repair cartilage defects, and poor integration is one of the reasons that leading to failure repairing. Chondrocytes migration capability is proven to have correlation with integration and some pathways, such as Src-phosphorylated phospholipase Cγ1-extracellular regulated kinase 1/2 has been confirmed to have correlation with the migration ability of chondrocytes, but the correlation with the integration is still unknown.
OBJECTIVE: To determine the chondrocyte signaling pathways involved in autologous chondrocyte migration and their effects on cartilage integration in autologous chondrocyte implantation.
METHODS: Articular chondrocytes were isolated from immature pig knee joints. The cells were divided into four groups: Src group, phosphorylated phospholipase Cγ1 group, extracellular regulated kinase 1/2 group and control group, then the Boyden chambers were used to quantify the chondrocyte migration. The chondrocytes/cartilage ring integration model was developed and cultured for 28 days, and then histology, biochemistry, biomechanics, western blot analysis and cell tracking analysis were performed to observe the differences between the control group and the suppression groups.
RESULTS AND CONCLUSION: The migration ability of chondrocytes was significantly decreased after pretreated with inhibitors. After the chondrocytes/cartilage ring co-cultured for 28 days, Western blot analysis showed that the pathway inhibitors has been presented in the entire culture cycle. The number and length of chondrocytes migrated into the integration area, collagen secretion level, matrix and mechanical strength in the control group were higher than those in three suppression groups. The results suggest that chondrocyte migration ability can affect the cartilage integration capability through Src-phosphorylated phospholipase Cγ1-extracellular regulating kinase 1/2 signal transduction pathway.

Key words: chondrocytes, transplantation, autologous, immunoblotting, biomechanics

CLC Number:

R617

Lu Yi-ming, Gui Jian-chao, Xu Yang, Yin Zhao-wei, Yang Xiao-fei, Jiang Yi-qiu. Correlation between chondrocyte migration and integration capability during autologous chondrocyte transplantation[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(44): 7721-7728.

Figures/Tables 5

在倒置显微镜下观察博伊登小室后发现，化学趋化因子(血清)能促进Src-磷酸化磷脂酶Cγ1-细胞外调节蛋白激酶1/2。当Src、磷酸化磷脂酶Cγ1、细胞外调节蛋白激酶1/2受到抑制后，软骨细胞的迁徙能力下降，表现为每个视野下迁徙过膜的软骨细胞数量减少(P < 0.05)，见图2。结果说明在化学趋化因子的刺激下Src、磷酸化磷脂酶Cγ1、细胞外调节蛋白激酶1/2和软骨细胞的迁徙具有相关性。 2.2 Src、磷酸化磷脂酶Cγ1和细胞外调节蛋白激酶1/2在体外整合模型中同样影响软骨细胞的迁徙能力 Src、磷酸化磷脂酶Cγ1、细胞外调节蛋白激酶1/2已经被证明在博依登小室中影响软骨细胞的迁徙能力，但在体外整合模型中是否发挥作用还不能确定。实验结果显示种植在软骨环中的软骨细胞具有迁徙的能力。此外，它们可以穿过整合界面，迁徙到宿主软骨中去，见图3。迁徙细胞的迁徙能力，反映为最远迁徙距离以及最大迁徙细胞数，抑制组是明显低于正常组的，表现为正常组的最远迁徙距离以及迁徙细胞数显著高于其他3个抑制组(P < 0.05)，见图3。结果证明了Src、磷酸化磷脂酶Cγ1、细胞外调节蛋白激酶1/2信号通路在类似于自体细胞软骨移植的整合模型中对细胞迁徙具有重要意义。 2.3 软骨整合受到Src、磷酸化磷脂酶Cγ1和细胞外调节蛋白激酶1/2介导的迁徙通路的影响在Src、磷酸化磷脂酶Cγ1和细胞外调节蛋白激酶1/2没有受到抑制的情况下，软骨细胞的迁徙能力被证明是增强的。但是迁徙能力增强到底对整合有着何种效果还不得而知。如图4中所示，在没有抑制剂存在的情况下，更多的软骨细胞迁徙到整合区域中(苏木精-伊红染色)。这些软骨细胞保持正常的表型正如图中所示分泌较多的Ⅱ型胶原和较少的Ⅰ型胶原。如图中番红O和Ⅱ型胶原的染色，正常组中在整合区域要比其他3个抑制组要分泌更多的细胞外基质和胶原。而生物力学的结果是与组织学染色结果密切相关的。整合区域中正常组的DNA含量要其他3个抑制组明显增多，见图5，结果提示更高的细胞密度存在于整合区域中。整个内芯的DNA含量并没有太大的区别，见图5A，所以能认定抑制剂对细胞增殖并没有太大的意义。因此判断正常组整合区域中的细胞较其他3个抑制剂组多是因为细胞迁徙的原因，而不是细胞增殖的原因。整个内芯中的糖胺多糖/DNA或者胶原/ DNA差异并无显著性学意义，见图5，说明抑制剂对细胞的分化并没有影响。正常组迁徙到整合区域的细胞较多，导致分泌的胶原和细胞外基质也比抑制组高，整合效果因此更好，继而导致生物力学明显要强于其他3个抑制组。"

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