Multilayered Engineered Tissue Sheets for Vascularized Tissue Regeneration

Abstract

A major hurdle in engineering thick and laminated tissues such as skin is how to vascularize the tissue. This study introduces a promising strategy for generating multi-layering engineered tissue sheets consisting of fibroblasts and endothelial cells co-seeded on highly micro-fibrous, biodegradable polycaprolactone membrane. Analysis of the conditions for induction of the vessels in vivo showed that addition of endothelial cell sheets into the laminated structure increases the number of incorporated cells and promotes primitive endothelial vessel growth. In vivo analysis of 11-layered constructs showed that seeding a high number of endothelial cells resulted in better cell survival and vascularization 4 weeks after implantation. Within one week after implantation in vivo, red blood cells were detected in the middle section of three-layered engineered tissue sheets composed of polycaprolactone/collagen membranes. Our engineered tissue sheets have several advantages, such as easy handling for cell seeding, manipulation by stacking each layer, a flexible number of cells for next-step applications and versatile tissue regeneration, and automated thick tissue generation with proper vascularization.

In Vivo Observation of Endothelial Cell-Assisted Vascularization in Pancreatic Cancer Xenograft Engineering

“…In contrast, the EHCH group showed homogeneous tissue formation, with the adenocarcinoma cells presumably surviving the early necrotic period due to less severe oxygen deficiency because of the dispersed cancer cell colonies. These results indicate that early connection to the host circulation system is essential for cell survival and cancerous xenograft tissue growth [37,38]. Furthermore, success in xenograft engineering also depends on limited oxygen deficiency in first implant stages.…”

Embossed Membranes with Vascular Patterns Guide Vascularization in a 3D Tissue Model

“…An embossed electrospun membrane was created using a vacuum forming method on PCL (PCL, Sigma, MO, USA) electrospun membranes. The PCL membrane was prepared as in a previous study [6]. A 12.5% ( w / v ) solution of PCL (80,000 M W ) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, BioPrince, Seoul, Korea) was dissolved with stirring at 80 °C for at least 4 h. The PCL solution was loaded into a 10 mL syringe that was fitted with a 25-gauge blunt needle tip.…”

“…After removal from the vacuum forming chamber, membranes were bonded to a frame for layering, which was used in a previous study [6,15]. Frames were fabricated with a 10 × 10 mm 2 square window with a polyethylene terephthalate (PET) sheet by Sahm-Oh Precision Machining (Seoul, Korea).…”

“…We defined regions of interest (ROIs, transplanted embossed tissue region) in three immunofluorescence figures for every group and obtained the area fraction for each ROI and each molecule (Ang-1) [18,19,20] to indicate their expression levels per frame analyzed. We also defined ROIs for each fluorescent area and divided them by the DAPI ROI area to obtain the percent area data and the area fraction [6,20].…”

“…To generate 3D tissue substitutes, we tried to stack up to 11 electrospun membranes; both sides of the polycaprolactone (PCL) electrospun membrane were seeded with endothelial cells and fibroblasts. These were then implanted into a subcutaneous pocket in a mouse [6]. This process resulted in vascularization four weeks after implantation.…”