Macroporous hydrogels formed by high internal phase emulsion-templating and their applications in tissue engineering

摘要

This thesis reports and discusses the development of several multifunctional biopolymers to be used in a cleaner approach for producing high porosity macroporous polymers by high internal phase emulsion (HIPE) templating for application as tissue engineering scaffolds. The aim is to identify and create biocompatible polymers that can self-stabilize HIPEs, be self-crosslinkable and also act simultaneously as the matrix for the macroporous polymers after emulsification, solidification and removal of the templating phase and aqueous solvent for the polymers. udFirstly, the design of a self-emulsifying biopolymer was carried out using a chitosan based biopolymer grafted with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and oligolysine (CSNLYS). Self-emulsification of HIPEs was found to be successful using this copolymer. The HIPEs could be solidified by raising the temperature above the lower critical solution temperature (LCST) of the PNIPAM component to 40°C. In addition, it was found that by changing the degree of polymerization of the grafted oligolysine, HIPEs with very different emulsion droplet sizes resulted, ranging from microemulsions with average droplet sizes of 0.13 µm, to macroemulsions with average droplet sizes of 10.5 µm. Polymerized (high internal phase macroemulsion) (polyHIPE) and polymerized (high internal phase 7 microemulsion) (polyHIPME) were formed by removing the liquid templating phases resulting in closed celled high porosity foams. udChitosan-graft-poly(N-isopropylacrylamide)-graft-oligoproline (CSN-PRO) and chitosan-graft-poly(N-isopropylacrylamide)-graft-oligo(glutamic acid) (CSN-GLU) were synthesized next to produce self-stabilized HIPE. CSN-PRO was found to be able to stabilize HIPE but not CSN-GLU, forming closed pores with pore sizes ranging from 32 µm to 71 µm. Upon addition of a low concentration of the surfactant PEG(20)sorbitan monolaurate, and varying the polymer concentration and internal phase volume ratio, different polyHIPEs with pore size of up to 143 µm, porosities of up to 99%, surface areas >300 m 2 /g and controlled pore interconnectivity can be formed. The CSN-PRO stabilized polyHIPEs are able to retain their thermoresponsiveness and remain intact when immersed into water at physiological temperature but dissolve below their LCST, which is useful in applications such as drug delivery and for tissue engineering scaffolds. Murine embryonic stem cells which are nonanchorage dependent were seeded to assess biocompatibility and were found to be able to survive and enter the pores of the poly(CSN-PRO)HIPE hydrogel. udTo produce scaffolds suitable for the attachment of anchorage dependent cells, a polypeptide, gelatin was used to create the self-emulsifying copolymer gelatin-graft-PNIPAM (GN). It was found GN does self-stabilize HIPEs. Upon solidification of the HIPEs and the removal of the templating oil phase and water from the aqueous phase highly porous and interconnected tissue 8 engineering scaffold resulted without the use of any additional surfactant. Poly(GN)HIPEs can be formed by two different solidification mechanisms as conferred by the components of GN, gelatin and PNIPAM. By inheriting the temperature sensitivity of these two components, GNHIPEs can be solidified at either 4°C due to the gelatin component or 40°C due to the PNIPAM component. The physical properties of the resultant self-stabilized poly(GN)HIPEs can be controlled by varying the aqueous phase, emulsion phase volume ratio and solidification temperature. Because of the inherent temperature sensitivity, poly(GN)HIPE hydrogels are able to response rapidly to changes in temperature during the initial cell culturing period. Fibroblast cells seeded into the scaffold were seen to thrive, spread and proliferate in a culture period of 10 days, with a maximum depth of penetration of 360 µm. The cell-laden poly(GN)HIPE scaffold was shown to be injectable through a syringe without harming the encapsulated cells. This system provides a new strategy for the easy fabrication of safe and injectable biocompatible scaffolds for tissue engineering.