Development of three-dimensional patterning strategies for osteochondral tissue engineering

Fully-realised three-dimensional patterning strategies enable the development of heterogeneous constructs which can recreate tissue architecture and cellular microenvironments over a large range of length scales. This in turn allows the development of more effective tissue models and tissue engineering therapies. The work presented in this thesis was designed to address the development of patterning methodologies and compatible biomaterial formulations. Poly(lactic-co-glycolic acid)-based (PLGA-based) microspheres were utilised for temporally-controlled protein delivery. Robust protocols were developed for the production of microspheres with two different mean sizes to provide distinct release kinetics which could be further tailored by the addition of a PLGA-poly(ethylene glycol)-PLGA (PLGA-PEG-PLGA) triblock copolymer. A semi-automated microinjection/micromanipulation (MM) system was used to precisely position individual microspheres into cell culture substrates. This approach has the potential to replicate complex interacting signal environments as seen in developmental and repair processes. Demineralised bovine bone, processed with or without a decellularisation step, was enzymatically digested to form solutions capable of gelation under physiological conditions. The resulting hydrogels outperformed collagen as in vitro culture substrates for bone-derived cells and are promising injectable scaffold materials. They were also formed into beads which could encapsulate exogenous proteins and which may be utilised in MM-based patterning strategies. Bioplotting was used to produce alginate hydrogel constructs containing highly viable cell populations. This technique was also used to deposit a PLGA-PEG microparticulate material which could be sintered under physiological conditions to achieve bone appropriate mechanical properties. PLGA-PEG/alginate dual material constructs could also be produced incorporating independent patterns of these two materials and of two cell populations and two protein signals. Bioplotting could therefore be used to produce sophisticated tissue engineering constructs for the repair of large, complex defects. Though this work focused on osteochondral applications much of the data is also more widely-applicable

Development of three-dimensional patterning strategies for osteochondral tissue engineering

Fully-realised three-dimensional patterning strategies enable the development of heterogeneous constructs which can recreate tissue architecture and cellular microenvironments over a large range of length scales. This in turn allows the development of more effective tissue models and tissue engineering therapies. The work presented in this thesis was designed to address the development of patterning methodologies and compatible biomaterial formulations. Poly(lactic-co-glycolic acid)-based (PLGA-based) microspheres were utilised for temporally-controlled protein delivery. Robust protocols were developed for the production of microspheres with two different mean sizes to provide distinct release kinetics which could be further tailored by the addition of a PLGA-poly(ethylene glycol)-PLGA (PLGA-PEG-PLGA) triblock copolymer. A semi-automated microinjection/micromanipulation (MM) system was used to precisely position individual microspheres into cell culture substrates. This approach has the potential to replicate complex interacting signal environments as seen in developmental and repair processes. Demineralised bovine bone, processed with or without a decellularisation step, was enzymatically digested to form solutions capable of gelation under physiological conditions. The resulting hydrogels outperformed collagen as in vitro culture substrates for bone-derived cells and are promising injectable scaffold materials. They were also formed into beads which could encapsulate exogenous proteins and which may be utilised in MM-based patterning strategies. Bioplotting was used to produce alginate hydrogel constructs containing highly viable cell populations. This technique was also used to deposit a PLGA-PEG microparticulate material which could be sintered under physiological conditions to achieve bone appropriate mechanical properties. PLGA-PEG/alginate dual material constructs could also be produced incorporating independent patterns of these two materials and of two cell populations and two protein signals. Bioplotting could therefore be used to produce sophisticated tissue engineering constructs for the repair of large, complex defects. Though this work focused on osteochondral applications much of the data is also more widely-applicable