Electrospun polymer mesh material, a potential hiding place for bacteria causing infection?

Introduction: Electrospinning has gained widespread interest in the field of tissue engineering as a method for producing porous non-woven meshes with nanometer scale fibers. The structure of electrospun meshes with inherently high surface to volume ratio can improve cell attachment, enhance drug loading capacity, and realize sustained and controlled drug delivery for devices such as prosthetic heart valves and surgical meshes. However, the high porosity of electrospun meshes may preferentially enhance colonization by bacteria. The small size of bacteria enables them to reach remote surfaces of the mesh which are less accessible to e.g. host tissue cells or immune cells. The interstitial spaces (pores) between electrospun fibers can be nano- to macrometers in size. Pore size generally decreases with smaller fiber diameters. Especially meshes with small pores can effectively filter bacteria from tissue cells, thus forcing the tissue cells to initially colonize only the surface of the mesh while the bacteria may penetrate the mesh and colonize its interior. To test the hypothesis that electrospun meshes with small pore size predispose for bacterial colonization we developed an in vitro model for studying bacterial infiltration in electrospun polycaprolactone (PCL) meshes with different fiber diameters. For the optimization of the model electrospun PLA/PCL mesh was used.

Methods: PLA/PCL mesh and PCL meshes with large and small fiber diameters were electrospun using chloroform, chloroform/pyridinium formiate or chloroform/methanol as the solvent. To produce fluorescent PCL fibers, coumarin 102 was added to the PCL polymer solution. The fiber diameter of the different PCL meshes was analyzed by scanning electron microscopy. After disinfection with 70% ethanol, meshes were incubated for 1.5h with Staphylococcus aureus bacteria expressing the red fluorescent mCherry protein. PLA/PCL and PCL meshes with infiltrated bacteria were fixed with paraformaldehyde and analyzed using a Leica SP8 confocal laser scanning microscope.

Results and Discussion : PLA/PCL mesh had an approximate average fiber diameter of 0.3µm. Four

different PCL meshes were produced with average fiber diameters of 6µm, 3µm, 2.5µm and <1µm. Optimization experiments with PLA/PCL showed that it was possible to visualize the mesh by confocal microscopy using laser reflection, however with low resolution. Infiltration of bacteria throughout this mesh was observed. Preliminary confocal microscopy results showed that incorporation of coumarin 102 in electrospun PCL made it possible to visualize single fibers and reconstruct a three-dimensional (3D) image of the full thickness of an electrospun mesh with infiltrating bacteria (figure 1). Future experiments need to address whether pore size forms a potential risk of bacterial infection because of inaccessibility to host phagocytic cells.


Figure 1: PCL mesh (6 µm fibers) analyzed with confocal microscope. A topview (upper pictures), a side view of full thickness of the mesh (bottom pictures). Blue fluorescent fibers (left);. PCL mesh with infiltrating bacteria, a greyscale image (right); bacteria are depicted as bright small spheres and present in al depths of the mesh