Electrospun chitosan membranes have been investigated for guided bone regeneration but are susceptible to swelling, dissolution, and loss of biomimetic nanofiber structure due to residual acid salts. A novel process was investigated for acidic salt removal from chitosan electrospun in 70% trifluoroacetic acid (TFA) by treating with triethylamine (TEA)/acetone and di-tert-butyl dicarbonate (tBOC) instead of the common NaCO treatment. TFA salt removal and nanofiber structure stabilization were confirmed by EDS, FTIR, F NMR and electron microscopy before and after soaking in water. Membrane degradation after 4 weeks in PBS with 100 µg ml lysozyme and osteoblastic proliferation were similar between TEA/tBOC-treated and NaCO-treated membranes. A simulated surgical tear test using surgical tacks showed that the peak tensile tear strength of the TEA/tBOC-treated chitosan membranes (62.1 ± 1.9 N mm) was significantly greater than a commercial polylactic acid (PLA) membrane (13.4 ± 0.4 N mm), similar to one commercial collagen membrane (55.3 ± 7.5 N mm) but lower than another commercial collagen membrane (133.9 ± 21.5 N mm). Rat 8 mm critical-sized calvarial defects covered with TEA/tBOC-treated chitosan membranes prevented soft tissue infiltration and supported new bone growth (15.76 ± 10.28%) similar to a commercial collagen membrane (16.08 ± 10.69%) at 12 weeks based on microCT analyses. Hence our novel TEA/tBOC process significantly improved nanofiber structure and mechanical strengths of electrospun chitosan membranes as compared to NaCO treated membranes, without affecting in vitro degradation or cytocompatibility, improved membrane mechanical properties to be greater than a commercial PLA membrane and to be in range of commercial collagen membranes and supported calvarial bone defect healing similar to collagen. Thus TEA/tBOC-treated chitosan membranes exhibit many characteristics and properties that strongly support their potential for use in guided bone regeneration.
The use of chitosan based nanofiber membranes in guided bone regeneration (GBR) is limited by its uncontrolled swelling and mechanical instability in aqueous environments. This paper describes the significantly improved stability and properties of surface butyrylated chitosan nanofiber (BCSNF) membranes that greatly enhance their potential in GBR. The BCSNF membranes exhibited an overall degree of substitution of 1.61, an average diameter of 99.3 ± 33.7 nm, and a 75% decrease in swelling with an approximate doubling in suture pull out strengths as compared to unmodified fibers in aqueous environment. In a five week phosphate-buffered saline-lysozyme degradation study, it was found that the remaining mass fraction of BCSNF membranes was 11.5% more than that of unmodified fibers. In vitro, the BCSNF membranes were found to support the adhesion and proliferation of fibroblasts and were cell occulusive. In vivo, the BCSNF membranes were found to significantly improve the regeneration of a rat calvarial critical size defect in a 12 week healing period and showed better barrier function than commercially available collagen membranes with little soft tissue penetration through the membranes. Taken together, these data provide strong scientific evidence for use of BCSNF membranes in GBR applications.
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