Effect of Multiple Confined Compression/Stress Relaxation Cycles on Cross-linked Alginate Pore Size

Materials and Methods: Solutions of 0.5 mg/ml FITC-Dextran MW markers (66 kDa/5.8 nm ,500 kDa/14.5 nm) dissolved in 0.9% (w/v) NaCl (saline) were prepared. Microcaspules (MC)s made of 2% (w/v) alginate formed by polyelectrolyte complexation were extruded from a 32 G needle into a 1% (w/v) calcium chloride solution and cross-linked for 60 min. After saline washes, the 3.5 mm MCs embedded in 2 mls of fluorescent marker were subjected to up to five consecutive confined compression/stress relaxation cycles under a 12.7 mm flat indenter using the Mach-1 mechanical testing systems (Biomomentum Inc.) for correlating the effect of mechanical treatment to fluorescent marker convection/diffusion under the following protocol: 28% compression under a load of 10 kg at a constant strain rate of 0.5 mm/s, hold time of 5 s, and then drop to preload. Image acquisition was conducted under the microscope at seven radial positions along the perimeter of the capsule using an FITC/Acridine Orange filter followed by processing using ImageJ (version 1.49). The average radial fluorescent (R) distance was used as a metric to monitor the progress of diffusion after each compression. Empty uncompressed control microcapsules were incubated in the MW markers and a two sample t-test was used at the 95% confidence interval using MATLAB (v2016) to test the difference in radial diffusion between the compressed (experimental) and control MCs.

Results and Discussion: As indicated by the p-values (<0.05) presented in Table 1, there is a significant difference at the 95% confidence interval between the experimental MCs and the controls after the third mechanical cycle. A sampled difference is illustrated graphically for the 500 kDa marker in Figure 1a where the radial distance traveled is significantly higher for the experimental capsule [Rexperimental= 467 µm± 33 µm] as compared to control [Rcontrol=324 µm ± 11 µm]. After the third compression, there is also a marked decrease in the Young’s Modulus (E) (EN2= 57 kPa and EN3= 16 kPa) suggesting mechanical weakening due to breaking of the cross-links with no leakage in the supernatant as shown in Figure 1b. As a result of this decrease in elasticity, the membrane has become more permeable to the 500 kDa/ 14.5 nm marker.