Crosslinking And Performance Evaluation Of Chitosan-Based Nanofibers For Tissue Scaffolding

In recent years, fabrication of nano-to micro-fibrous scaffolds to biologically mimic the native extracellular matrix, via an emerging technique termed electrospinning, has drawn a great deal of attention in the tissue engineering community. As one of the most popular scaffolding materials, the natural biopolymer chitosan (CTS) has been electrospun into nanofibers via different routes. This was achieved by electrospinning of pure chitosan dissolved in specialty solvents (e.g., trifluoroacetic acid (TFA)) or by electrospinning fiber-foming-agents-doped chitosan blends. However, regardless of the routes employed, these electrospun CTS nanofibers are poor in water resistance by becoming swelled and/or being dissolved away while contacting the water, which would affect the fiber morphology and performance in applications. Also, the weak mechanical performance in wet condition is another well-known problem. To address these noted issues, major objectives of the present research work are:1) To optimize the electrospinnability of CTS;2) To chemically crosslink the electrospun CTS nanofibers by using a naturally occurring crosslinking agent genipin, and to throroghly characterize the physical, chemical, and biological properties of the CTS nanofibers after crosslinking treatment;3) To prepare chitosan based hydroxyapatite (HAp) scaffold in nanofibrous form so as to investigate its osteogenic capabilities after crosslinking with genipin.The electrospinnability of chitosan, when using ultra-high molecular weight polyethylene oxide (PEO) as the fiber forming agent, was firstly studied. It was found that when the mass ratios of CTS/PEO were85/15,90/10,95/5, and97/3, all these mixed solutions can be electrospun into nanofibers with good fibrous morphology, even though the amount of fiber forming agent PEO has been reduced down to3%only. With the electrospun CTS, we determined the mass ratios of CTS to PEO through elemental analysis and X-ray photoelectron spectroscopy, and studied the molecular interaction between them through the FTIR method. The above prepared electrospun nanofibers are of water sensitivity to some content. But, as expected the CTS nanofibers containing the minimum amount of PEO (3%) possessed improved stability of fiber morphology in wet condition. Mechanical testing results showed that when the mass ratio of CTS/PEO was95:5in hybrid nanofibers, the mechanical performance of the nanofibrous mat is preferable in drying regime with a tensile strength up to18.96±1.62MPa. However, the tensile strength decreased significantly down to a level of merely6.39±0.95MPa in wet condition.To alleviate the above noted problems and take the biological compatibility into consideration, a naturally occurring crosslinker genipin was employed to have the electrospun CTS nanofibers chemically crosslinked, in comparison with the commonly used crosslinker glutaraldehyde. Physical, chemical and biological performance of the CTS nanofibers after crosslinking was subsequantly examined. The SEM results showed that both genipin and glutaraldehyde were beneficial to retain the nanofibrous morphology of the electrospun CTS under wet condition, with the efficacy relevant to concentration of the crosslinking agent and crosslinking time. FTIR spectroscopy revealed the chemical crosslinking nature between genipin and CTS. XRD results showed that, after crosslinking with genipin, crystallinity of chitosan nanofibers was increased, which interprets the noted declines in swelling degree and enzymatic degradation rate of crosslinked CTS nanofibers. There were obviously differeneces in nanofiber morphology between the crosslinked and non-crosslinked CTS nanofibers after soaking in phosphate buffered saline (PBS, pH=7.2) for72h. Also, the morphology of crosslinked CTS nanofibers can be well maintained after being subjected to lysozyme degradation for3weeks. Furthermore, culturing L929fibroblasts on the CTS nanofibrous scaffolds crosslinked by varied concentrations of genipin indicated that crosslinking via genipin promoted cell proliferation compared to the non-crosslinked counterparts, and we found that the most suitable concentration of genipin solution is0.5%. Longer time cell culturing demonstrated that CTS nanofibrous scaffolds crosslinked with genipin can be more beneficial to cellular growth than that of the ones crosslinked with glutaraldehyde.Moreover, we prepared chitosan based hydroxyapatite nanocomposites nanofibers HAp/CTS to mimic the nanostructure of the natural bone, and performed the steps to have the electrospun HAp/CTS nanofibers chemically crosslinked based on the above developed crosslinking method. Subsequantly, using bone marrow derived mesenchymal stem cells (BMSCs) as the seeding cells, we examined osteogenesis of the HAp/CTS nanofibers after crosslinking. Likewise, the SEM results showed that crosslinking treatment was beneficial to retain the nanofibrous morphology of the electrospun HAp/CTS. BMSCs were able to adhere to and proliferate on the genipin crosslinked HAp/CTS nanofibrous scaffolds, indicating good cellular biocompatibility. Furthermore, expression of the bone-associated protein alkaline phosphatase (ALP) confirmed that the genipin crosslinked HAp/CTS composite nanofibers promoted BMSCs differentiation toward osteogenic lineage. All the results demonstrated that the crosslinking protocol developed from our current work is feasible to preserve the nanofibrous morphology of electrospun chitosan based fibrous materials with appropriate biocompatibility as cellular scaffolds for tissue engineering.