Chitosan

Example 12

There has been a growing interest in the fabrication of nanofibers derived from natural polymers due to their ability to mimic the structure and function of extracellular matrix. Electrospinning is a simple technique to obtain nano-micro fibers with customized fiber topology and composition (FIGS. 33A and 33B). The chitosan electrospun nanofibers have recently been extensively studied due to the favorable properties of chitosan such as controllable biodegradation, good biocompatibility and high mechanical strength. Currently, chitosan can be electrospun from a solution of chitosan dissolved in either trifluoroacetic acid (TFA) or acetic acid (HAc). However, processes to remove residual acid and acid salts from the electrospun material generally resulted in a swelling of fibers and deterioration of the nano-fibrous structure. Crosslinking in combination with neutralization methods also had not been effective at preventing loss of nano-fibrous structure.

The current study aimed to improve and maintain nano-fibrous and porous structure of the electrospun membranes by introducing a new post electrospinning chemical treatment. Membrane thickness was tripled in this research in order to increase the general tearing strength. Scanning electron micrograph (SEM) examination (FIG. 33C) and transmission electron micrograph (TEM) examination (FIG. 33D) showed Fiber diameters of the triethanolamine/N-tert-butoxycarbonyl (TEA/t-BoC) treated membranes ranged from 40 nm to 130 nm while fiber diameters were not able to be determined for the Na₂CO₃ group. Membranes treated by TEA/tboc (FIG. 34A) exhibited more nano-scale fibrous structure than membranes treated by saturated Na₂CO₃ (FIGS. 35B-35D, as seen demonstrated in scanning electron micrographs. After immersion in PBS for 24 hours, membranes treated by TEA/tboc exhibited less than 30% swelling (FIG. 34B) and retained their nanofibrous structure, compared with membranes treated by Na₂CO₃ (FIGS. 35B-35D) or compared with the non-treated chitosan membrane (FIG. 35A). After soaking the TEA/tBoc treated membranes in water overnight, membranes still kept the porous structure. In both, the before and after water status, fibers kept diameters in the nanometer range (FIG. 35C). TEA/tBoC modified nanofiber membranes also well preserved their fibrous structure over 4 weeks in physiological solution compared with Na₂CO₃ treated membranes (FIG. 35D).

Chitosan membranes treated by TEA/tboc showed better nano-fiber morphology characteristics than membranes neutralized by saturated Na₂CO₃ solution before and after being soaked in PBS. Retention of the nanofibrous structure for guided tissue regeneration applications may be of benefit for enabling nutrient exchange between soft gingival tissue and bone compartments and for mimicking the natural nanofibrillar components of the extracellular matrix during regeneration.

Example 5

To further explore the potential of the electrospun chitosan nanofibers in tissue engineering applications, osteoblast proliferation on the membrane of electrospun chitosan nanofibers was examined by Celltiter Glo Assay Kit. As shown in FIG. 12, there was no statistical difference in the day 5 growth of cells on chitosan compared with acylated (acetyl- and butyryl-) and deacylated electrospun chitosan nanofiber membranes, suggesting the electrospun chitosan nanofibers membranes were non-toxic.

The cell morphology on the materials was visualized with fluorescence microscope after osteoblast cells were cultured on the top of the materials for 5 days. The cells grown on the electrospun chitosan nanofibers showed characteristic shapes associated with osteoblast cells, such as elongated/stretched shape, suggesting the material did not interfere with the growth of the osteoblasts.

These surprising results suggested that the problems with dissolution and swelling observed with electrospun chitosan fiber membranes can be solved by the reversible acylation method. The mechanisms behind the process were elucidated based on the data obtained from the FTIR, XPS and SEM analyses. The acylation method could potentially be used to synthesize other modified chitoan nanofibrous material containing acyl moieties as well.

Example 6

FIG. 13 shows the in vitro degradation of the acetyl-chitosan, butyryl-chitosan and Na₂CO₃ treated chitosan film when exposed to 100 μg/ml of lysosome in phosphate buffered saline (PBS). Both acetyl-chitosan and butyryl-chitosan resisted degradation and maintained mass longer than Na₂CO₃ treated chitosan film.

FIG. 14 shows the ultimate suture pullout load for acetyl-chitosan, butyryl-chitosan and regenerated (deacylated) chitosan when wet or dry.

FIG. 15 shows the weight gain or swelling after exposure to a hydrophilic solvent of Na₂CO₃ treated chitosan film, acetyl-chitosan, butyryl-chitosan and regenerated (deacylated) chitosan. The stabilized electrospun chitosan fiber membranes show good cytocompatibility, structural stability, suggesting future exploration in tissue engineering applications.