Medium molecular weight chitosan

The extrusion solution is comprised of chitosan. Medium molecular weight chitosan with a deacetylation degree of 75–85% was purchased from Sigma Aldrich (Sydney, Australia) and used without further purification. Chitosan was found to be selective in the noncovalent wrapping of carbon tubes and specially disperse them over other impurities^{8 (link)}. The solution was prepared by dissolving of 3 g chitosan (Medium molecular weight chitosan with deacetylation degree of 75–85% (Sigma Aldrich)) in 100 ml acetic acid (1% v/v). The solution was stirred at room temperature for 6 h. Then, carbon tubes, either untreated or ultrasonicated, were dispersed in chitosan solution at a concentration of 50 wt%, per chitosan weight, with the aid of sonication in a low power ultrasonic bath. The dispersion was stored into a 5 ml syringe that was fixed on a steel micronozzle (0.6 mm in diameter). The extrusion process was performed using a bioplotter 3D printer on a 3 axis stage, where the motion was controlled using a preprogramed patterning procedure. The printing flow was controlled by the air pressure. The ink was printed (with the nozzle feed rate of 5 mm/s) and then allowed to coagulate in a non-solvent bath. The height of liquid in the coagulation bath was 1 cm above the printing materials. Finally, the printed materials were dried in air.

Chitosan medium molecular weight (190–310 kDa, deacetylation degree 75–85%) was purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium alginate was obtained from Himedia Co. (Mumbai, India). Carbon tetrachloride, thiobarbituric acid, and 5,5-dithiobis-2-nitrobenzoic acid were purchased from Sigma-Aldrich. Absolute alcohol, glacial acetic acid, and all other chemicals were of analytical grades and were used without further modification.

Calreticulin human recombinant protein was bought from Raybiotech, Inc. Norcross, GA, (United States). Anti-proliferating cell nuclear antigen (PCNA) antibody and anti-mouse IgG2a Fluorescein IsoTioCyanate (FITC) were bought from Santa Cruz Biotechnology, Dallas, TX, United States. Calreticulin BioAssayTM Enzyme-Linked ImmunoSorbent Assay (ELISA) kit (Human) was used. Chitosan medium molecular weight (degree of deacetylation 75–85%), gold (III) chloride hydrate, dimethyl sulfoxide (DMSO), 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI), phosphate buffer saline (PBS) and antibiotic-antimicotic solution were purchased from Sigma Aldrich, St. Louis, MO, United States. Thiazolyl blue tetrazolium bromide (MTT) was purchased from Affymetrix, Inc. Cleveland, OH, United States. Fetal bovine serum (FBS), Trypsin-EDTA (1x), and Dulbecco´s Modified Eagle medium (DMEM) were purchase from Gibco, Thermo Fisher Scientific, Waltham, MA, United States. De-ionized (DI) water (Milli-Q®, Water Purification System, Merck Millipore) was also used. Human keratinocyte cells (HaCaT), human umbilical vein endothelial cells (HUVEC) and mouse fibroblast cells (NIH/3T3) were purchased from ATCC, Manassas, VA, United States.