Irgacure 2959 photoinitiator

Hydrogel tubes were generated as previously described (Dumont et al., 2019 (link)). Briefly, 20% w/v 8-arm polyethylene glycol maleimide (PEG-MAL, 20 kDa; JenKem, Plano, TX) was crosslinked with 5 mM slow-degrading-plasmin-sensitive YKND cross-linking peptide (Ac-GCYKNDGCYKNDCG; Genscript, Piscataway, NJ) (Shikanov, Smith, Xu, Woodruff, & Shea, 2011 (link)) to form microspheres through water-oil emulsion with diameter ranging between 15 and 150 μm and an average of 45 μm. The PEG-YKND solution was homogenized in silicone oil (Fisher, Hampton, NH) with 2% Tween-20 (Sigma, St. Louis, MO) at a speed of 4000 rpm for 1 minute. Microspheres were rinsed by centrifugation three times. Irgacure 2959 photoinitiator (Sigma) dissolved in N-vinylpyrrolidinone (660 mg/mL; Sigma) was added to the microspheres at a final concentration of 1% w/v. The resulting microspheres were then packed into polydimethylsiloxane (PDMS, Dow Corning, Midland, MI) molds to generate PEG tubes (approximate OD: 600 μm, ID: 250 μm, porosity 66%) and exposed to an ultraviolet lamp for 3 minutes to initiate free radical polymerization. Tubes were rinsed three times, dehydrated, and stored at −80 until use. Tubes were cut to length during surgery to ensure fit within the defect.

Labeled cells (2 × 10⁷ cells/ml) were mixed with a
polymer solution of 10% w/v PEODA (Laysan Bio, Arab, AL), 0.05% w/v Irgacure
2959 photoinitiator (Sigma-Aldrich), and 2.5 mg/ml Hyaluronic Acid (1100
kDa; Lifecore Biomedical, Chaska, MN) in sterile PBS (Sharma et al., 2007 (link)). Next, the
cell-polymer solution was pipetted into plastic moulds and placed under a
long wave, 365-nm ultraviolet light at 4 mW/cm2 (Omincure S2000, Excelitas
Technologies, Mississauga, Ontario, Canada) for 5 min to induce
polymerisation and generate 5 × 3 mm cylindrical constructs. The
constructs were then transferred to chondrogenic medium in 24-well plates
for in vitro studies or implantation experiments.