组织工程椎间盘体内原位植入的实验研究

Tissue engineered intervertebral disc implantation in situ：an experimental study

中文关键词:  组织工程  椎间盘  体内原位植入  聚乳酸聚酯  犬

英文关键词:Tissue engineering  Intervertebral disc  In vivo  Poly L-lactic-co-glycolic acid  Canine

中文摘要:

  【摘要】 目的：观察组织工程椎间盘原位植入Beagle犬腰椎间盘后支架与种子细胞的转归及犬腰椎间盘的形态与功能变化。方法：应用聚乳酸聚酯（poly L-lactic-co-glycolic acid，PLGA）可吸收多孔支架复合犬髓核细胞构建组织工程椎间盘。将18只Beagle随机分为3组，均摘除L4/5椎间盘髓核，A组单纯髓核摘除，B组植入空白PLGA支架，C组植入组织工程椎间盘。术后定期行X线片及MRI检查，分别于术后4、8及12周处死动物行组织病理及免疫组化观察。结果：全部动物术后存活。4周后，X线片示3组椎间盘高度均有下降，C组下降幅度较小，与A、B组比较有显著性差异（P<0.05）；A组和B组手术节段屈伸活动度（ROM）明显大于C组（P<0.05）；MRI示A组与B组椎间盘退变明显，呈“黑间盘”改变，而C组椎间盘退变程度较轻，呈灰色，MRI T2加权像上椎间盘信号灰度值测量结果示A、B组明显低于C组（P<0.05）。术后4周B、C组组织学观察未见到支架材料结构，A、B组椎间盘内纤维组织增生形成瘢痕样组织，C组于术后8周仍能观察到植入的红色荧光细胞，有大量类软骨细胞增生伴丰富的细胞外基质。术后8周免疫组化显示三组均有Ⅰ、Ⅱ型胶原着色，其中C组Ⅱ型胶原染色强度高于Ⅰ型，A、B组Ⅰ型胶原染色强度高于Ⅱ型。结论：PLGA组织工程椎间盘支架在体内可降解吸收；种子细胞可存活并分泌Ⅰ、Ⅱ型胶原，能阻止纤维组织侵入及瘢痕形成；维持了脊柱节段稳定及生理活动功能。

英文摘要:

  【Abstract】 Objective：To construct tissue engineered poly L-lactic-co-glycolic acid（PLGA） scaffold-nucleus pulposus cells intervertebral disc and to investigate the in vivo performance of the composite and its morphology and function in a canine model.Method：Nucleus pulposus（NP） cells isolated from the lumbar intervertebral discs of Beagle dog were cultured and proliferated in vitro and seeded into a 3-D porous PLGA scaffold.The composite was tested in 18 beagle dogs which were randomly divided into 3 groups：nucleotomy alone（group A），nucleotomy with PLGA implantation（group B） and nucleotomy with PLGA scaffold/NP cells composite implantation（group C）.X-ray and MRI scan were performed before and after the operation.Evaluation of disc height，segment stability，biomechanics and immunohistochemical analysis were performed and their results were recorded.Result：All animals were alive after operation.4 weeks later，disc height decreased in all three groups，with group C less severe than group A and B（P<0.05）.Flexion-extension ROM of group A and B were higher than group C（P<0.05）.More remarkable disc degeneration was noted in group A and B which showed black disc than group C which showed gray disc on T2 weighted MRI with T2-weighted signal intensity of group A and B lower than group C（P<0.05）.No PLGA scaffolds structure was found at 4 weeks postoperatively，scar tissue formation was evidenced in group A and B，while PHK-26 positive cells were evidenced in group C 8 weeks later with amount of chondrocyte cells proliferation and matrix formation.Type Ⅰ and Ⅱ collagen were noted in all three groups 8 weeks later，and type Ⅱ collagen staining in group C was more severe than type Ⅰ，type Ⅰ collagen staining in group A and B was more severe than type Ⅱ.Conclusion：The NP cell-PLGA scaffold composite is degradable and the seed cells can release type Ⅰ and Ⅱ collagen which can prevent scar infiltration as well as reconstruction of segmental stability and alignment.