Guided tissue regeneration (GTR) membranes not only can hamper undesirable tissues down-growth into the defects but also can selectively promote the in-growth of regenerative bone tissue, playing a critical role in periodontal regeneration. Herein, a bi-layered electrospun membrane with different sized pores was designed and fabricated by adjusting electrospinning parameters combing with facile two-step electrospinning. The small-sized pore layer (SL) as occlusive layer consisted of electrospun poly (lactic-co-glycolic acid) (PLGA) nanofibers, while the macroporous osteoconductive layer (ML) was attained via introducing the nano-hydroxyapatite (nHA) particles into PLGA nanofibers during electrospinning. Morphological results such as surface topography, nanofiber size, and pore size distribution, showed that the SL exhibited a dense structure with pore size mainly from 4 to 7 μm. In contrast, the ML possessed a loosely packed structure with pore size mainly from 20 to 28 μm, which was beneficial to the infiltration of the cells. Fourier transform infrared spectroscopy (FTIR), Energy dispersive spectrometer (EDS), and X-ray diffractometry (XRD) results showed that nHA particles were evenly loaded in PLGA nanofibers. In vitro biodegradation tests suggested that the bi-layered membrane possessed a proper degradation timeframe, which must function for at least 4 to 6 weeks. The cell experiments indicated that the bi-layered electrospun membrane possessed good cytocompatibility and proved the effective barrier potency of the small-sized pore layer. Furthermore, as revealed by the alkaline phosphate activity test, the PLGA/nHA layer possessed an improved osteogenic capacity for Human osteosarcoma cells (MG63). These results indicate that the bi-layered electrospun membrane may have potential for periodontal tissue regeneration.
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