Developing a Biodegradable Electrospun Membrane as a Periosteum Substitute for Critical Size Bone Defect Repair

Critical size bone defects (CSBD) are too large (>1-2 cm) for spontaneous repair. Here, a new concept of a ‘hybrid graft’ CSBD reconstruction was investigated, whereby a barrier membrane to contain graft material and support periosteal regrowth was developed alongside examination into periosteum and bone marrow (BM) as mesenchymal stem cell (MSC) sources. To create a ‘bioinspired’ substitute membrane, human and porcine periosteum samples were analysed for thickness and tissue architecture. Free surface electrospun membranes containing poly(ε-caprolactone) (PCL) (M3%-PCL) and collagen-PCL (M6%-CP70:30) were developed and characterised, followed by barrier function testing and MSC attachment and proliferation. ‘Clinically relevant’ human periosteum samples generally lacked a cambium layer, but were positive for CD271, an MSC marker throughout the collagenous fibrous layer. Porcine periosteum was variable in thickness (127-2310 μm). MSCs were significantly more frequent in periosteum (2% vs 0.0008%, P=0.004), but formed significantly smaller colonies in vitro 6.1 mm2 vs 15.5 mm2, P=0.0006), compared to BM MSCs. Whilst osteogenic and chondrogenic potential was similar, periosteum MSCs were significantly less adipogenic. Live cell imaging showed that BM MSCs were more migratory than periosteum MSCs in early culture (<3 weeks). M3%-PCL and M6%-CP70:30 were made of nano-scale randomly aligned fibres, forming small pore diameters (mean, 1.4 μm and 0.4 μm respectively). M6%-CP70:30 supported significantly lower (P<0.01) MSC attachment and proliferation than M3%-PCL, potentially due to a ‘burst’ release of collagen. Significant spontaneous alignment of MSCs on M3%-PCL was seen over 4 weeks. Spontaneous MSC migration through both membranes was prevented for up to a week in vitro. In summary, two free surface electrospun membranes have been developed with the potential for supporting, to different extents, MSC proliferation, whilst acting as a barrier to cell migration, suitable for CSBD repair. Periosteum was shown to be at least equivalent and possibly, superior source of MSCs to BM for membrane colonisation.