Many biomaterial scaffolds have been developed for use in tissue engineering usually for populating with a single cell-type. In this study we demonstrate the production of bilayer and trilayer nanofibrous/ microfibrous biodegradable scaffolds suitable for the support, proliferation and yet segregation of different tissueshere used to separate soft tissue from bone forming tissue and keratinocytes from fibroblasts. Essentially we describe a nanofibre barrier membrane which is permeable to nutrients coupled with attached microfibers (either on one side or both sides) to support the proliferation of different cell types either side but prevents migration of cells across the barrier. Such membranes would be suitable for guided tissue regeneration in areas where one wishes to support both soft and hard tissues but keep them separated. We describe a sterile bilayer membrane electrospun from polyhydroxybutyrate-co-hydroxyvalerate (PHBV) (nanofibres) and polylactic acid (PLA) or poly ε-caprolactone (PCL) (microfibres) and a trilayer membrane electrospun in layers of PLA, PHBV, then PLA. These membranes are biocompatible, biodegradable and capable of supporting two different cell populations.

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.(1, 2, 3) Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes--here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications. Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA). Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes--one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.(4) An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

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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.