Prolonged in vitro precultivation alleviates post-implantation in ammation and promotes stable subcutaneous cartilage formation in a goat model
Prolonged in vitro precultivation alleviates post-implantation in ammation and promotes stable subcutaneous cartilage formation in a goat model
复制标题
长时间的体外预培养可减轻山羊模型中的植入后炎症并促进稳定的皮下软骨形成
作者：
Yi Liu
合成的生物可降解支架如聚乳酸涂覆的聚乙醇酸（PLA-PGA）特别适用于工程化成形软骨如耳廓，但它们诱导严重的炎症反应，特别是在免疫侵袭性皮下部位，工程化自体软骨的再吸收。我们之前在兔模型中的研究已经证明，体外预培养2周可以显著减轻PLA-PGA工程软骨移植物诱导的植入后炎症，但在临床前山羊模型中未能再现该结果。本研究的目的是调查是否延长在体外预培养可以形成一个成熟的软骨移植物，以抵抗急性宿主反应，并促进稳定的皮下软骨形成在临床前山羊模型。将山羊软骨细胞接种到PLA-PGA支架上，体外预培养2、4、8和12周，然后植入自体山羊皮下1和8周。对体外工程化软骨（vitro-EC）进行组织学检查（苏木精和伊红，番红-O，胶原II）。对1周的外植体进行组织学检查，并对CD 3、CD 68、胶原I和细胞凋亡进行染色。通过组织学、湿重、体积、糖胺聚糖（GAG）定量和杨氏模量评价8周外植体。随着体外培养时间的延长，体外EC的质量得到改善，支架残留量减少;在体外预培养较长时间的外植体中观察到更明显的软骨形成，免疫细胞（CD3和CD68阳性）较少，凋亡细胞和I型胶原表达较少。皮下再生的新软骨在长期植入后变得更加成熟。这些结果表明，延长在体外预培养允许形成一个成熟的软骨移植物，以抵抗急性宿主反应，促进稳定的自体山羊皮下软骨形成。这些发现可为耳廓、气管、鼻、眼睑等软骨的工程化提供参考。
Synthetic biodegradable scaffolds such as polylactic acid coated polyglycolic acid (PLA-PGA).are especially suitable for engineering shaped cartilage such as auricle, but they induce a serious inflammatory reaction particularly in the immunologically aggressive subcutaneous site, leading.to resorption of the engineered autologous cartilage. Our previous study in a rabbit model has demonstrated 2 weeks of in vitro precultivation could significantly alleviate the post-implantation inflammation induced by PLA-PGA engineered cartilaginous grafts, but reproduction of this result failed in a preclinical goat model. The aims of the current study were to investigate whether prolonged in vitro precultivation could form a mature cartilaginous graft to resist the acute host response and promote stable subcutaneous cartilage formation in a preclinical goat model. Goat chondrocytes were seeded onto PLA-PGA scaffolds, in vitro precultivated for 2, 4, 8, and 12 weeks, and then implanted subcutaneously in autologous goats for 1 and 8 weeks. The in vitro engineered cartilage (vitro-EC) was examined histologically (hematoxylin and eosin, safranin-O, collagen II). The 1 week explants were examined histologically and stained for CD3, CD68, collagen I, and apoptosis. The 8 week explants were evaluated by histology, wet weight, volume, glycosaminoglycan (GAG) quantification and Young’s modulus. With prolonged in vitro time, the quality of vitro-EC improved and the amount of scaffold residue decreased; more pronounced cartilage formation with fewer immune cells (CD3 and CD68 positive), apoptotic cells, and less collagen I expression were observed in explants that had been in vitro precultivated for a longer period. The subcutaneously regenerated neocartilage became more mature after prolonged implantation. These results suggested that prolonged in vitro precultivation allowed formation of a mature cartilaginous graft to resist the acute host response and promoted stable subcutaneous cartilage formation in autologous goats. These findings may provide useful reference for engineering auricle, trachea, nose, and eyelid shaped cartilage, for example.