An Antimicrobial Peptide-Loaded Gelatin/Chitosan Nanofibrous Membrane Fabricated by Sequential Layer-by-Layer Electrospinning and Electrospraying Techniques

Guided bone regeneration (GBR) technique is widely used in the treatment of bone defects caused by peri-implantitis, periodontal disease, etc. However, the GBR membranes commonly used in clinical treatments currently have no antibacterial activity. Therefore, in this study, sequential layer-by-layer electrospinning and electrospraying techniques were utilized to prepare a gelatin (Gln) and chitosan (CS) composite GBR membrane containing hydroxyapatite nanoparticles (nHAp) and antimicrobial peptide (Pac-525)-loaded PLGA microspheres (AMP@PLGA-MS), which was supposed to have osteogenic and antibacterial activities. The scanning electron microscope (SEM) observation showed that the morphology of the nanofibers and microspheres could be successfully produced. The diameters of the electrospun fibers with and without nHAp were 359 +/- 174 nm and 409 +/- 197 nm, respectively, and the mechanical properties of the membrane were measured according to the tensile stress-strain curve. Both the involvement of nHAp and the chemical crosslinking were able to enhance their tensile strength. In vitro cell culture of rat bone marrow mesenchymal stem cells (rBMSCs) indicated that the Gln/CS composite membrane had an ideal biocompatibility with good cell adhesion, spreading, and proliferation. In addition, the Gln/CS membrane containing nHAp could promote osteogenic differentiation of rBMSCs. Furthermore, according to the in vitro drug release assay and antibacterial experiments, the composite GBR membrane containing AMP@PLGA-MS exhibited a long-term sustained release of Pac-525, which had bactericidal activity within one week and antibacterial activity for up to one month against two kinds of bacteria, S. aureus and E. coli. Our results suggest that the antimicrobial peptide-loaded Gln/CS composite membrane (AMP@PLGA-MS@Gln/CS/nHAp) has a great promise in bone generation-related applications for the unique functions of guiding bone regeneration and inhibiting bacterial infection as well.