OBJECTIVES The utilization of viral vectors to deliver genes of interest directly to meniscus cells and promote long-term modulation of gene expression may prove useful to enhance meniscus repair and regeneration. The objective of this study was to optimize and compare the potential of lentivirus (LV) and adeno-associated virus (AAV) to deliver transgenes to meniscus cells in both intact meniscus tissue and isolated primary cells in monolayer. DESIGN Porcine meniscus tissue explants and primary meniscus cells in monolayer were transduced with LV or self-complementary AAV2 (scAAV2) encoding green fluorescent protein (GFP). Following transduction, explants were enzymatically digested to isolate meniscus cells, and monolayer cells were trypsinized. Isolated cells were analyzed by flow cytometry to determine percent transduction. RESULTS LV and scAAV2 showed a high transduction efficiency in monolayer meniscus cells. scAAV2 was most effective at transducing cells within intact meniscus tissue but the efficiency was less than 20%. Outer zone meniscus cells were more readily transduced by both LV and scAAV2 than the inner zone cells. Higher virus titers and higher cell density resulted in improved transduction efficiency. Polybrene was necessary for the highest transduction efficiency with LV, but it reduced scAAV2 transduction. CONCLUSIONS Both LV and scAAV2 efficiently transduce primary meniscus cells but only scAAV2 can modestly transduce cells embedded in meniscus tissue. This work lays the foundation for viral gene transfer to be utilized to deliver bioactive transgenes or gene editing machinery, which can induce long-term and tunable expression of therapeutic proteins from tissue-engineered constructs for meniscus repair and regeneration.

中文翻译：

---

使用慢病毒和腺相关病毒优化半月板细胞转导用于基因编辑和组织工程应用。

目的利用病毒载体将感兴趣的基因直接递送至半月板细胞并促进基因表达的长期调节可能有助于增强半月板修复和再生。本研究的目的是优化和比较慢病毒 (LV) 和腺相关病毒 (AAV) 将转基因递送至完整半月板组织和分离的单层原代细胞中的半月板细胞的潜力。设计 猪半月板组织外植体和单层原代半月板细胞用 LV 或编码绿色荧光蛋白 (GFP) 的自互补 AAV2 (scAAV2) 转导。转导后，外植体被酶消化以分离半月板细胞，单层细胞被胰蛋白酶消化。通过流式细胞术分析分离的细胞以确定转导百分比。结果 LV 和 scAAV2 在单层半月板细胞中显示出高转导效率。scAAV2 在完整的半月板组织内转导细胞最有效，但效率低于 20%。与内区细胞相比，外区半月板细胞更容易被 LV 和 scAAV2 转导。更高的病毒滴度和更高的细胞密度导致转导效率提高。聚凝胺是 LV 最高转导效率所必需的，但它降低了 scAAV2 转导。结论 LV 和 scAAV2 都能有效地转导原代半月板细胞，但只有 scAAV2 可以适度转导嵌入半月板组织的细胞。这项工作为病毒基因转移用于传递生物活性转基因或基因编辑机制奠定了基础，