2.1. Interpenetrating Polymer Network (IPN) Synthesis

Interpenetrating polymer network hydrogels were made with a final rat tail collagen type I (COL-I) (Corning, Bedford, MA, USA) concentration of 1.5 mg/mL mixed in a 1:1 ratio with either a final concentration of 5 (interpenetrating polymer network (IPN) 5), 10 (IPN 10), or 15 (IPN 15) mg/mL of ultrapure, low-viscosity, and high glucuronic acid (LVG) alginate (ALG) (Novamatrix, Sandvika, Norway) in phosphate buffered saline (PBS) (Thermo Fisher Scientific, Carlsbad, CA, USA). According to the manufacturer’s instructions, collagen was brought to alkalinity to assist in the gelling procedure. The alginate stock solution was first centrifuged at 650 g for 5 min, diluted to 10, 20, and 30 mg/mL, mixed with the collagen solution and added to custom-made molds. These molds were then placed in petri dishes for collagen gelation at 37 °C for 30 min. In the meantime, 20 mM calcium chloride (Sigma, Arklow, Ireland) in ultrapure water was prepared and brought up to a pH of 7.2 using 1 N NaOH. The calcium chloride was used to ionically crosslink the alginate present in the IPN hydrogels for an additional 3 h at 37 °C. Control sample hydrogels were made that completely consisted of 1.5 mg/mL collagen type I by following the same gelation procedure as outlined above. The samples for compressive testing were stored in ultrapure water whereas the samples for histology and scanning electron microscopy (SEM) were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 4 h, subjected to 30 min of 30% ethanol (EtOH) and stored in 50% EtOH.