| ObjectiveGuided bone regeneration as an effective bone augmentation technique has been widely used in the treatment of implant patients suffering from alveolar bone defects. In this study, chitosan and collagen blend solutions in acetic acid is mixed to develop nanofiber menbranes with electospinning technique. Biocompatibility of biomimetic nanostructure membrane, also physical and biological properties are evaluated. It is also assessed that the positive effect of nanostructure membranes and lyophilized films synergistically used to guided bone-defect regeneration in vivo.Method1). Different ratios of chitosan/collagen/PEO spinning solutions were prepared and divided into three major groups:pure PEO, chitosan/PEO, chitosan/collagen/PEO. The spinnability of all solutions is studied in order to fabricate chitosan-collagen nanofiber membranes by electrospinning.2).The nanofiber membrane needs to be further crosslinked by glutaraldehyde vapor to improve its structural stability in case of degradation or fracture in water.3). The structure and composition of the nanofiber membrane was analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), respectively; Cytotoxicity assays and cell growth and morphology observation on nanofibers are prepared to evaluate film’s biocompatibility.4). The chitosan and collagen blend solutions are lyophilized in vacuum to form microporous membranes after being frozen at4℃for12h,-20℃for24h,-80℃for24h. The freezed-dried film was treated by pressure at room temperature to improve its tensile strength.5). The bilayer composition membrane with the layer of nanofiber film closed to defects while the layer of freezed-dried film above it, Bio-gide membrane, freezed-dried film, no membrane, are randomly applied in circular calvarial critical-sized defects (d=5mm) in Sprague Dawley rats. At6weeks, all rats were sacrificed and histology was performed. The samples’paraffin sections were stained with haematoxylin-eosin and Masson Trichrome for histological evaluation and histomorphometric analysis. Bone regeneration ratio was measured and compared among four groups.Results1). When PEO concentration at3.5%, chitosan concentration at1%and collagen concentration at0-6%, bead-free and randomly arrayed nanofibers could be obtained by electrospinning. It is feasible to fabricate a nanostructure membrane with a certain thickness through continuously electrospinning for4-5h.2) After crosslinking by glutaraldehyde vapor, the nanofiber membrane became visibly yellowish and shrunk slightly. Water-resistant test confirmed it’s insoluble and stable in water.3). MTT test confirmed the nanofiber membrane had no cytotoxicity to MC3T3-E1. After seeding12h and36h, MC3T3-E1cells spread well on the surface of the nanofiber and cells both interact and integrate well with the surrounding fibers.4). After mechanical pressing treatment, the tensile strength of lyophilized film remarkably increased from0.69±0.28MPa to11.9±2.9MPa, meanwhile, the elastic modulus of the film improved to192.2±39MPa.5). In vivo study showed that bilayer composition membranes were available for successful regeneration of bone and bone regeneration ratio reached43.1±1.49%, which is similar to the Bio-gide membrane group of41.36±2.6%.ConclusionThe chitosan-collagen nanofiber membrane can be fabricated by electrospinning. It has good biocompatibility and its nanostructure contributes to cells absorption and proliferation. The bilayer composition membrane has adequate mechanical strength to maintain stable space and ability of promoting new bone regeneration. |
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