Tissue Engineering (TE) aims to devise solutions for the healing of critical defects which cannot heal naturally, within the host tissue. In a typical TE approach, biodegradable scaffolds are used to fill the defect site to provide temporary mechanical support and to serve as a three-dimensional substrate for cell attachment and proliferation. These TE scaffolds need to have a highly interconnected porous architecture to enable cell infiltration, nutrient flow, and integration of the material within the host tissue. To date, various scaffold fabrication routes have been reported. However, recently, emulsion templating has gained particular attention as a scaffold fabrication technique due to its ability to introduce (i) up to 99% porosity, (ii) high interconnectivity, and high tunability. The technique is briefly based on the preparation of emulsion composed of at least two immiscible liquids where one phase is dispersed in the other phase and solidification of the continuous phase of the emulsion and removal of the internal phase. Fabrication of emulsion templated matrices made of a wide range of synthetic and natural polymers have been reported. However, polycaprolactone (PCL)-based emulsion templated substrates have previously been shown to be challenging to formulate due to the high viscosity of the polymer, which limits the efficient mixing of the two phases within the emulsion.
In this study, tetramethacrylate functionalised PCL (4PCLMA) was synthesised, and photocurable PCL-based Polymerised High Internal Phase Emulsions (PolyHIPEs) were developed. The effect of diluting solvent volume and composition on the stability of HIPEs and morphology of PolyHIPEs was studied in depth. Following the development of the formulation of PCL PolyHIPEs using a solvent blend of chloroform and toluene and their initial cytotoxicity test were conducted. Then, the suitability of PCL PolyHIPEs to be used as a dental membrane (BM) was tested. Bilayer BM was developed by combining electrospinning and emulsion templating techniques. Impact of air plasma treatment of PolyHIPEs on cell viability and cell penetration was investigated. Results showed that PCL electrospun layer was capable of limiting cell infiltration at least for four weeks while the morphology of the PCL PolyHIPEs allows infiltration of bone cells through the pores. Especially, cell infiltration was significantly higher in air plasma treated PolyHIPEs. Ex ovo chick chorioallantoic membrane (CAM) assay showed that the pore structure of PolyHIPEs allows blood vessel growth through the pores.
PCL PolyHIPE-based multiscale porous scaffolds were also fabricated by combining emulsion templating with additive manufacturing. In this study, PCL-based emulsions were prepared and transferred into the syringe of the pneumatic extrusion printer, and scaffolds were printed and cured simultaneously by the integrated LED of the printer. This multi-step fabrication route is a promising way to develop scaffolds with more complex shapes using three-dimensional data of the defect site. To increase the biological performance of the polymeric multiscale porous scaffolds, they were decorated with in vitro generated bone extracellular matrix (ECM). Briefly, bone cells were grown on PCL PolyHIPEs, and a decellularisation procedure was applied to remove the genetic material and create a biohybrid scaffold. The presence of bone ECM, which is mainly composed of collagen and mineral, was shown to improve the osteogenic performance of PolyHIPE scaffolds in vitro and enhance the angiogenic performance in vivo. In addition, a higher degree of cell infiltration and a higher number of blood vessels within the macropores was observed in biohybrid scaffolds compared to PolyHIPEs.
To summarise, 4PCLMA was used as a novel biomaterial with an emulsion templating as a scaffold fabrication technique, to produce highly tunable scaffolds, demonstrating that emulsion templated 4PCLMA is a promising candidate to be used for tissue engineering applications.
