Aim 2: Design and characterize model biomaterials to assess the foreign body response in vivo.
4.1. Design criteria
The criteria for this specific aim is to determine the concentration of GA required to crosslink the gelatin hydrogel that would result in hydrogel properties that are
statistically different such as the stiffness. The crosslinked hydrogels were characterized by four different properties in order to determined significant differences between the hydrogels. The four characterization properties include mechanical stiffness, degradation, swelling, and crosslinking density.
4.2 Methods
4.2.1. Preparation of glutaraldehyde and glycine
Glutaraldehyde (25% stock solution) from Sigma Aldrich was diluted with phosphate buffer saline (PBS) to 0.05%, 0.1%, and 0.3% concentrations. 0.1 M glycine (Sigma Aldrich) was made by dissolving 0.225 g of glycine in 30 mL of sterile PBS. The glycine solution was then sterilized via heating and UV light for at least 2 hours.
4.2.2 Preparation of sodium citrate and sodium phosphate
Sodium citrate (Sigma Aldrich) was made by dissolving 2.94 g of sodium citrate tribasic in 1000 mL of DI water. The pH was of the solution was adjusted to 6.0 by adding approximately 200 uL of 37% hydrochloric acid. 0.5 mL of Tween-20 (Sigma
Aldrich) was added to the solution and mixed well. Sodium phosphate (Sigma Aldrich) was made by dissolving 1.2 g of sodium phosphate monohydrate in 1000 mL of PBS and adding 0.1 mL of Tween-20.
4.2.3. Preparation of crosslinked gelatin hydrogels
10 wt% gelatin was made by heating and dissolving 4 g of gelatin (Sigma Aldrich, Type B bovine skin) in 40 ml of PBS. 8 mL of the gelatin solution was transferred into petri dishes and allowed to cool to room temperature. The petri dishes were placed in a refrigerator or ice bucket in order for the gelatin to completely solidify. Once solidified, a 5 mm biopsy punch was used to punch the gelatin into cylindrical disks. The gelatin hydrogels were crosslinked by immersion in 0.05%, 0.1%, and 0.3% GA solutions in a shaker overnight. The crosslinked gelatin hydrogels were then
sterilized under UV light for at least 2 hours. The hydrogels were then washed 4 times in PBS for 15 minutes each wash. Afterwards, the hydrogels were put into 0.1M glycine solution in a shaker overnight to neutralize any residual GA. The hydrogels were washed an additional two times in PBS for 15 minutes each. Again, the hydrogels were sterilized under UV light for at least 2 hours.
4.2.4 Hydrogel characterization
4.2.4.a Mechanical testing and Young’s modulus of hydrogels
The crosslinked gelatin hydrogels were mechanically tested via compression tests in order to determine the effects of crosslinking. The force and displacement data were
obtained via Bose Electroforce 3100 Test Instrument of Drexel University for each of the hydrogel samples. Using a caliper, the dimensions, the height and the diameter, of the hydrogels were measured. The hydrogels were compressed to 30% strain at a rate of 0.333 per second. The stress and strain were calculated, and the slope of the initial linear region, about 5-7% strain, was used to determine the Young’s modulus.
4.2.4.b Degradation of gelatin hydrogels
Crosslinked hydrogels were immersed for one week in collagenase (Sigma Aldrich) to monitor enzymatic degradation. The concentration of collagenase was 5 ug/mL. The initial mass of the hydrogels were measured, and then the hydrogels were massed every 24 hours after blotting dry on a paper towel to determine the percent mass loss over time.
4.2.4.c Swelling ratio of hydrogel
The swelling ratio of the hydrogels was calculated by the following equation:
(20)
where ws is the swollen weight and wd is the dry weight. The swollen weight was
calculated by measuring the mass of the hydrogels after allowing them to reach
equilibrium after swelling in PBS overnight. The hydrogels were then allowed to dry at room temperature in order to measure the dry weight.
4.2.4.d Crosslinking density of hydrogels
The crosslinking density of each hydrogel was calculated by the modified Flory Rehner equation [72]:
[ ( ) ]
(21)
where ε is the crosslinking density, vp is the polymer volume fraction, χ is the polymer-
solvent interaction parameter, ρ is the density of the polymer, and V1 is the molar volume
of solvent. vp was calculated by the measuring the dimensions of the hydrogels. χ was
determined via literature search [72]. The density of the hydrogel was calculated by measuring the mass of the hydrogel divided by the volume of the hydrogel. V1 was
calculated by determining the molar mass of H2O. The crosslinking density was
calculated with the given parameters from experiments (vp, ρ) and literature.
4.2.5 Statistical analysis
Statistical analysis was performed in MATLAB using one-way ANOVA with Tukey post hoc analysis in order to determine statistical significance between the crosslinked hydrogels.
4.3 Results
4.3.1 In vitro hydrogel characterization
The crosslinked gelatin hydrogels were characterized by mechanical testing, degradation, swelling, and crosslinking density. As seen in the figures below, the values
obtained for the hydrogels of the three different crosslinking concentrations were statistically different from each other in each of the experiments.
Figure 8: The crosslinked hydrogels characterized by the hydrogel properties. A. The crosslinking density of each hydrogel is significantly different. B. The stiffness of each hydrogel is significantly different. C. The 0.3% hydrogel was significantly different from the other two hydrogels. D. The degradation of each hydrogels were significantly different from day 7 to day 13. * indicates statistical significance (p<0.05) between the indicated groups. ** denotes p<0.01, *** denotes p<0.005, **** denotes p<0.001
A B
As expected, the crosslinking density of the hydrogels increased as the concentration of GA increased. From the ANOVA analysis, the three hydrogels (0.5%, 0.1%, and 0.3%) were statistically significant from each other with p < 0.05. Similarly, the Young’s modulus of the hydrogels increased with increasing crosslinking, and the hydrogels were all significantly significant from each other. The swelling ratio of the hydrogels
decreased with increasing crosslinking, which has been shown before [72]. With a network of crosslinks, hydrogels cannot absorb as much water compared to less
crosslinked hydrogels, thus reducing the swelling of the hydrogels. The swelling ratio of both the 0.05% and 0.1% crosslinked hydrogel was significantly lower than that of the 0.3% crosslinked hydrogel. The 0.05% and 0.1% hydrogels were statistically similar. In addition, the rate at which the hydrogels degraded decreased with increasing crosslinking. The hydrogel crosslinked at a higher concentration resulted in a slower degradation because of more crosslinks and bond formed within the network structure.