Production and Characterization of a Novel, Electrospun, Tri-Layer Polycaprolactone Membrane for the Segregated Co-Culture of Bone and Soft Tissue.

Sasima Puwanun,Moira Ireland,Nicola Green,Sheila Macneil,Gwendolen Reilly,Frazer Bye

doi:10.3390/polym8060221

Sasima Puwanun, Moira Ireland + Show 4 more

Open Access

https://doi.org/10.3390/polym8060221

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Journal: Polymers	Publication Date: Jun 7, 2016
Citations: 27	License type: CC BY 4.0

Affiliation: Naresuan University, University of Sheffield, Insigneo

Abstract

Composite tissue-engineered constructs combining bone and soft tissue have applications in regenerative medicine, particularly dentistry. This study generated a tri-layer, electrospun, poly-ε-caprolactone membrane, with two microfiber layers separated by a layer of nanofibers, for the spatially segregated culture of mesenchymal progenitor cells (MPCs) and fibroblasts. The two cell types were seeded on either side, and cell proliferation and spatial organization were investigated over several weeks. Calcium deposition by MPCs was detected using xylenol orange (XO) and the separation between fibroblasts and the calcified matrix was visualized by confocal laser scanning microscopy. SEM confirmed that the scaffold consisted of two layers of micron-diameter fibers with a thin layer of nano-diameter fibers in-between. Complete separation of cell types was maintained and calcified matrix was observed on only one side of the membrane. This novel tri-layer membrane is capable of supporting the formation of a bilayer of calcified and non-calcified connective tissue.

Highlights

An important clinical goal in tissue engineering is to move beyond the reconstruction of single tissue types to generate composite tissues, composed of two or more tissue types, which more closely reflect the basic unit for healing in vivo
Scaffolds were fabricated with an electrospinning rig, at room temperature, as previously described [1,5], with one rotating drum collector (126 mm diameter) and two syringe pumps, each with four needles, located on either side of the collector
For co-culture experiments without the barrier layer fibroblasts were labelled with CellTrackerTM Green as above and Human embryonic cell derived mesenchymal progenitor cells (hES-MPs) with CellTrackerTM Red (Invitrogen, Paisley, UK) prior to incorporation into the scaffold

Summary

Introduction

An important clinical goal in tissue engineering is to move beyond the reconstruction of single tissue types to generate composite tissues, composed of two or more tissue types, which more closely reflect the basic unit for healing in vivo. This is of particular significance in several clinical settings, including cleft lip/cleft palate repair, trauma surgery and tumor removal. The second was to seed the tri-layer scaffold with cells capable of generating bone and soft tissue, demonstrate localized mineral deposition and evaluate the extent to which the scaffold could maintain spatial separation of the two layers

PCL Scaffold Fabrication

Assessment of Cell Viability of hES-MPs on Electrospun PCL Scaffold

PCL Tri-Layer Scaffold Fabrication

Co-Culture between hBMSCs and onexperiments

Conclusions