In vitro and in vivo evaluations of a novel post-electrospinning treatment to improve the fibrous structure of chitosan membranes for guided bone regeneration.

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 Na2CO3 treatment. TFA salt removal and nanofiber structure stabilization were confirmed by EDS, FTIR, 19F NMR and electron microscopy before and after soaking in water. Membrane degradation after 4 weeks in PBS with 100 µg ml-1 lysozyme and osteoblastic proliferation were similar between TEA/tBOC-treated and Na2CO3-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-1) was significantly greater than a commercial polylactic acid (PLA) membrane (13.4  ±  0.4 N mm-1), similar to one commercial collagen membrane (55.3  ±  7.5 N mm-1) but lower than another commercial collagen membrane (133.9  ±  21.5 N mm-1). 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 Na2CO3 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.