The Feinstein Institutes for Medical Research, Manhasset, NY, USA; Division of Pediatric Otolaryngology, Steven and Alexandra Cohen Children's Medical Center, New Hyde Park, NY, USA.
The Feinstein Institutes for Medical Research, Manhasset, NY, USA.
Int J Pediatr Otorhinolaryngol. 2022 Apr;155:111066. doi: 10.1016/j.ijporl.2022.111066. Epub 2022 Feb 14.
To optimize a 3D printed tissue-engineered tracheal construct using a combined in vitro and a two-stage in vivo technique.
A 3D-CAD (Computer-aided Design) template was created; rabbit chondrocytes were harvested and cultured. A Makerbot Replicator™ 2x was used to print a polycaprolactone (PCL) scaffold which was then combined with a bio-ink and the previously harvested chondrocytes. In vitro: Cell viability was performed by live/dead assay using Calcein A/Ethidium. Gene expression was performed using quantitative real-time PCR for the following genes: Collagen Type I and type II, Sox-9, and Aggrecan. In vivo: Surgical implantation occurred in two stages: 1) Index procedure: construct was implanted within a pocket in the strap muscles for 21 days and, 2) Final surgery: construct with vascularized pedicle was rotated into a segmental tracheal defect for 3 or 6 weeks. Following euthanasia, the construct and native trachea were explanted and evaluated.
In vitro: After 14 days in culture the constructs showed >80% viable cells. Collagen type II and sox-9 were overexpressed in the construct from day 2 and by day 14 all genes were overexpressed when compared to chondrocytes in monolayer.
By day 21 (immediately before the rotation), cartilage formation could be seen surrounding all the constructs. Mature cartilage was observed in the grafts after 6 or 9 weeks in vivo.
This two-stage approach for implanting a 3D printed tissue-engineered tracheal replacement construct has been optimized to yield a high-quality, printable segment with cellular growth and viability both in vitro and in vivo.
使用体外结合两阶段体内技术优化 3D 打印组织工程气管构建体。
创建 3D-CAD（计算机辅助设计）模板；收获和培养兔软骨细胞。使用 Makerbot Replicator™2x 打印聚己内酯（PCL）支架，然后将其与生物墨水和先前收获的软骨细胞结合。体外：使用 Calcein A/Ethidium 进行死活测定以进行细胞活力测定。使用定量实时 PCR 进行以下基因的基因表达：I 型和 II 型胶原、Sox-9 和 Aggrecan。体内：手术分两阶段进行植入：1）索引程序：构建体植入颈阔肌袋中 21 天，2）最终手术：带有血管化蒂的构建体旋转到节段性气管缺损 3 或 6 周。安乐死后，取出构建体和天然气管进行评估。
体外：培养 14 天后，构建体显示出>80%的存活细胞。第 2 天，构建体中 II 型胶原和 Sox-9 过度表达，第 14 天，与单层软骨细胞相比，所有基因均过度表达。
第 21 天（在旋转之前），可以看到所有构建体周围都形成了软骨。在体内 6 或 9 周后，在移植物中观察到成熟软骨。
这种用于植入 3D 打印组织工程气管替代构建体的两阶段方法已得到优化，可产生高质量、可打印的节段，具有体外和体内细胞生长和活力。
