Hyaluronan Gel Crosslinking

Hyaluronan (HA) gel crosslinking was performed at 4 °C and at a constant HA: ADH: EDCI: HCl ratio. 25 mg of adipic dihydrazide (ADH) was dissolved into 100 mL of dH₂O and added to 20 ml of 1 % high molecular weight hyaluronan (Clear Solutions Biotechnology, Inc., Stony Brook, NY) in a 30 ml syringe. At this point in some preparations FN in PBS was added as needed to achieve a 30 mg/ml concentration to produce HA crosslinked to FN as well as to itself (HAFN). The mixture was stirred well, but slowly, to avoid excessive shear in the HA. 30 mg of 1-(3-dimethylaminoproyl)-3-ethylcarbodiimide hydrochloride (EDCI) was dissolved into 100 mL of dH₂O and added to the HA solution. The solution was stirred well and allowed to sit at 4 °C for four additional hours to allow for more complete homogeneity through diffusion. The HA solution was then degassed under a vacuum in an aspirator to remove small air bubbles and dissolved gas which may later come out of solution. This allowed maximal crosslinking as the reaction did not become disrupted by air bubbles. 115 mL of 1N HCl was added to the solution to achieve optimal crosslinking pH, and mixed in. The solution was allowed to crosslink for 24 hours at 4 °C.

Hyaluronan Gel Dialysis

Spectrum Spectra/Por Molecularporous Membrane 2 (MW 12,000-14,000 Da, Rancho Dominguez, California) dialysis tubing was prepared according to protocol. 7 inch strips were cut and soaked in sterile dH₂O for 30 minutes. The tubing was filled to 66% of capacity, leaving a small air pocket at one end of the tube. Both ends of tube were clamped and the tube soaked in 1 L dH₂O per 10 ml HA gel, with gentle mixing with magnetic stirbar. Crosslinked HAFN was dialyzed for three days at 4 °C, changing dH₂O twice a day. Complete FN retention onto the HA scaffold (a measure of intermolecular crosslinking) and lack of FN degradation due to crosslinking procedure was confirmed through Western blot analysis of the HAFN construct. Western blot was performed using a mouse- derived monoclonal antibody against the III8 of human FN (Lab Vision Corp., Fremont, CA) and HRP-goat-anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as a secondary antibody. Pure crosslinked HA was dialyzed by alternating 50 % ethanol solution with dH₂O for 2 days, then was dialyzed in dH₂O for 1 additional day, changing the water three times. For some experiments, free FN was added to crosslinked HA (HA+FN) by mixing 1mg/ml FN solution in PBS into previously crosslinked and dialyzed HA to a 30 mg/ml concentration.