Histological characteristics of the reconstructed anterior cruciate ligament with repetitive freeze-thawing allogenic Achilles tendon

doi:10.3969/j.issn.2095-4344.2017.16.015

Abstract

Abstract:

BACKGROUND: Thawed allografts are usually discarded for various reasons. Whether these discarded allografts can be refrozen for later use and their histological changes in vivo have not been reported.
OBJECTIVE: To investigate the histological characteristics of anterior cruciate ligament (ACL) reconstructed with the repetitive freeze-thawing allogenic Achilles tendon in New Zealand white rabbits.
METHODS: Allogenic Achilles tendons were harvested from adult male New Zealand white rabbits, and were stored at -8 ℃ and thawed at 20 ℃ for 1, 2, 3 and 10 times, respectively, after sealed package and 60Co irradiation. Twenty-four adult male New Zealand white rabbits were enrolled: the left and right knees of 12 rabbits were respectively reconstructed with 1 (control group) and 2 times of freeze-thawing allogenic Achilles tendon, and another 12 rabbits underwent reconstruction with 3 and 10 times of freeze-thawing allogenic Achilles tendon, respectively. Three specimens from each group were evaluated with modified histology grading scores at 6, 12 and 24 weeks to assess the cell morphology, cell quantity, matrix staining intensity, fibrocartilage formation, new bone formation, tendon healing and cartilage injury.
RESULTS AND CONCLUSION: The cell morphology, matrix staining intensity and total scores of the 10 times group were significantly higher than those of the other groups at 6 weeks (P < 0.05), but other parameters showed no significant differences among groups (P > 0.05). ACL reconstructed with 10 times of repetitive freeze-thawing allogenic Achilles tendons had higher histological scores at 6 weeks after modeling, but no significant differences were shown at 12 and 24 weeks after modeling. To conclude, our study only testifies better histological scores on the multiple times of freeze- thawing Achilles tendon than the less times of freeze-thawing Achilles tendon at the early period after operation.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Freezing, Histology, Anterior Cruciate Ligament, Tissue Engineering

CLC Number:

R318

Tian Ming, Yu Jia-kuo, Wu Yan-ping. Histological characteristics of the reconstructed anterior cruciate ligament with repetitive freeze-thawing allogenic Achilles tendon[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(16): 2546-2551.

Figures/Tables 2

2.1 实验动物数量分析 24只兔随机分成2组，实验过程无脱失，进入结果分析24只。 2.2 大体评估造模后6周：伤口愈合良好，兔膝关节活动度正常。重建的前交叉韧带与胫骨和股骨骨道口处可见部分愈合，并形成一定的张力，膝关节未见明显关节积液，可见滑膜中度增生，部分滑膜覆盖重建的前交叉韧带组织，滑膜表面可见少量充血，软骨外观形态尚可。重建的前交叉韧带部分被吸收，较正常前交叉韧带细。冻融10次组在粗细程度上稍粗于其他组(图2a)。造模后12周：重建的前交叉韧带与胫骨个股骨愈合良好，重建的前交叉韧带较粗壮，张力良好，颜色接近正常的前交叉韧带，周围滑膜增生减轻，呈轻度增生，未见明显关节积液，膝关节软骨轻度退变，不同冻融次数的各组间无明显差别(图2b)。造模后24周：重建的前交叉韧带与骨道外口愈合良好，张力较好，形态接近正常前交叉韧带，并有正常前交叉韧带的光泽，膝关节未见明显关节积液和滑膜增生，关节软骨退变较12周时严重，未见不同冻融次数的各组间有明显差别(图2c)。 2.3 组织学评估造模后6周：移植物周围有少量炎性细胞浸润，以淋巴细胞为主。移植物内出现大量细胞增殖数量甚至超过正常前交叉韧带，分布不均，在移植物边缘可以发现细胞集群并有血管增生，移植物中央细胞减少甚至发现无细胞区，冻融10次组的细胞分布较其他组均匀，细胞形态稍好于其他组，细胞增殖数量多于其他组但是无显著性差异，基质染色强度冻融10次组强于其他组，未见明显的软骨化区域，50%的腱骨界面区域有肉芽组织和纤维结缔组织与骨道壁紧密相连，局部仅有少量Sharpey样纤维连接形成，但未见明显的新骨形成。残留的Ⅰ型胶原仍然较多，有一定量的新生Ⅲ胶原分泌，未见明显的Ⅱ型胶原分泌(图3，4)。造模后12周：移植物内的细胞增殖数量仍然超过正常前交叉韧带，但是较造模后6周时有所下降，分布较造模后6周时均匀，大部分呈弥漫性增生，在移植物的边缘和中心仍然可见少量血管增生，大部分移植物的中央未发现明显的无细胞区，细胞分布较均匀，形态为卵圆形的成纤维细胞较多，与正常前交叉韧带相似的细长的梭形细胞较少，基质染色强度稍强于造模后6周，仅有很少的骨道边缘出现软骨化区域，区域内有少量Ⅱ型胶原表达，新骨形成仍然不明显，75%-50%的区域移植物与骨道之间有肉芽组织和纤维结缔组织紧密相连，有较多Sharpey样纤维连接，更成熟，更加致密有序，但是，造模后12周各组间对比未见明显差异。重建的前交叉韧带中有一定量的Ⅰ型胶原表达，有较多的Ⅲ型胶原分泌，未见明显的Ⅱ型胶原分泌(图5，6)。造模后24周：移植物中的细胞数量较造模后6，12周时明显减少，接近正常前交叉韧带的细胞数量。分布较12周时均匀，接近正常前交叉韧带，移植物中的血管数量较12周时减少，接近正常前交叉韧带，细胞逐渐由高代谢的卵圆形成纤维细胞转变为接近正常前交叉韧带的低代谢的细长梭形的细胞。基质染色强度与12周时差别不明显，腱骨界面出现大量纤维软骨形成，明显较12周时多，并有大量软骨细胞生成，区域内有大量Ⅱ型胶原表达，腱骨间由6周和12周时的间接止点愈合向直接止点愈合转变，部分骨道中有新骨形成，局部仍有 Sharpey样纤维连接较12周时更加致密有序，骨道中的大部分区域腱与骨的连接紧密，但是，术后24周的各组间未见明显差异。重建前交叉韧带中有大量Ⅰ型和Ⅲ型胶原分泌，未见明显的Ⅱ型胶原分泌(图7，8)。 2.4 组织学评分造模后6周冻融10次组的细胞形态，基质染色强度和总分明显高于其他组(P < 0.05)，其他各组评分差异无显著性意义(表2)。"

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