Haake mars modular advanced rheometer

Collagen gels were prepared as per the manufacturer's instructions with the pregelation procedure carried out on ice with cold-incubated pipette tips. Briefly, collagen (Collagen I heavy chain [HC], rat tail −0.02 N in Acetic Acid; BD Biosciences, Bedford, MA) was mixed with HBSS (10% v/v without Ca⁺⁺ or Mg⁺⁺), neutralized with sodium bicarbonate, and equilibrated to 5 mg/mL collagen in HBSS. Gelation was carried out at room temperature for 30 min, and the gels were further incubated at 37°C for a minimum of 1 h.
For measuring the physical property change, 280 μL collagen gels were incubated with 280 μL of 0 or 1 mM CeN for 30 min. After incubation, CeN solution was removed, and the gels were blot dried and incubated for 2 days at 37°C before the measurements. Oscillatory amplitude sweep with 1 to 100 Pa at an angular frequency of 1 Hz, as described by Evani et al,^{31 (link)} was used to calculate viscoelastic properties of the gels using the HAAKE MARS modular advanced rheometer (ThermoFisher Scientific, Inc.) equipped with parallel plate-PP20. Storage modulus in linear viscoelastic range was measured as an indicator stiffness (Fig. 2).

The elastic modulus was determined by using a mechanical tester (Hengyi, Shanghai, China). The samples were subjected to uniaxial compression testing at a steady strain rate of 1 mm/min and 0.1–5 Hz frequency at 25°C, and the elastic modulus was obtained by calculating the slope of the stress–strain curve for 0%–10% strain. Moreover, the original height of the sample before compression was recorded as H₀, and after the compression test, the final height of the sample was recorded as H_r. The ratio H_r/H₀ was used to measure the elasticity of the sample. The following rheological measurements were conducted at 37°C on a Haake Mars Modular Advanced Rheometer (Thermo Fisher Scientific, USA). At a fixed strain of 5%, a frequency sweep test was performed from 0.1 to 10 Hz to obtain the storage modulus (G′) and loss modulus (G″).
Next, 100 mg of the composite hydrogel from each group was immersed in 50 ml of PBS, and the drug release was analyzed in a shaker at 37°C. At predetermined time points, i.e., days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, 2 ml of the supernatant was collected and replaced with fresh PBS. The absorbance of the collected samples was measured by using a fluorescence spectrometer to evaluate the drug concentration and plot the in vitro drug release curve.