Development of a novel hollow fibre membrane for use as a tissue engineered bone graft scaffold

The most successful • therapy for repairing bone defects is currently the autograft. Autografts have several disadvantages associated with their use including a limited availability per patient, donor site morbidity and deformity, and the graft itself can be resorbed before osteogenesis is complete. Production of a synthetic bone graft, using tissue engineering principles, to overcome these drawbacks would be a significant step forward for orthopaedic surgery. The aim of this thesis was to develop a scaffold to address the mass transfer limitations of autografts and current synthetic scaffolds that are used for treating bone defects. A novel PLGA hollow fibre membrane scaffold was created using a poly(lactic-co-glycolic acid) N-methyl pyrrolidinone -water (PLGA-NMP-water) system by immersion precipitation. NMP was selected as the solvent due to the resulting asymmetric macroporous structure of the membrane that had interconnecting finger-like pores. The entire range of PLGA containing the racemic mix of PLLA and PDLA was found to be soluble in NMP with PDLLArPGA ratio 45:55 to 100:0. PLLA and PGA did not dissolve in NMP between 15-30°C, 10-40% (w/w). PLGA with the PLLA:PGA ratio of 75:25 (75L:25) was found to be soluble in NMP at 10% (w/w) across the temperature range tested. The hollow fibre membranes were spun using PDLLA:PGA ratios of 50:50 and 75:25 at 20 %(w/w) and 25 %(w/w) polymer concentrations, 0 mm and 30 mm air gaps, and 5.5 m/min and 7.7 m/min take-up rates. The structure was found to be optimal for use as a bone tissue engineering scaffold in the range of conditions tested when spun using a 20% (w/w) polymer solution with a zero air gap at a take up rate of 5.5 m/min. This resulted in a fibre with typical outer diameter of 700 pm and wall thickness of approximately 150 pm. The porous outer skin had pores of around 1 pm diameter for both 50:50 and 75:25. 50:50 was seen to have a relatively less dense outer skin at a given set of conditions. 70% ethanol was found to be a suitable sterilising agent, if not optimal due to its effect on the membrane morphology. It was also found that residual solvent could be reduced to 4 ppm after 7 days by soaking in water. The osteoblastic cell line 560pZIPv.neo (pZIP) and human bone derived cells (HBDC) were cultured on 100:0, 75:25, 50:50 and 75L:25 PLGA flat sheet membranes to assess the suitability of the membranes as a scaffold in comparison to tissue culture polystyrene (TCP). 75:25, 50:50 and 75L:25 were riot significantly different to the TCP (P<0.05) for both cells types during a 6 hour attachment period, 100:0 had significantly fewer HBDC after 6 hours but showed a comparable number of pZIP. After the 7-day proliferation study, the four membranes showed a similar number of pZIP to the TCP. 50:50 and 75:25 had significantly lower HBDC compared to TCP. HBDC showed osteogenic function (alkaline phosphatase activity and mineralisation) on all membranes after 3 weeks, but significantly less compared to TCP. 75:25 hollow fibre membranes were bedded into a hollow fibre module and seeded with pZIP. An in situ rotating seeding method was used over a 6 hour period and was found to be 4 times more efficient that traditional static seeding. The number of pZIP was significantly lower compared to the TCP control following a counter current fed perfusion set up for 7 days. The work in this thesis has shown that the PLGA hollow fibre membrane scaffold has the potential to address the current problems associated with autologous bone grafts and traditional tissue engineered scaffolds. A ck n ow ledg em ents