Photopolymerized Hydrogels for hMSC Encapsulation

Previously described methods were used to synthesize MeHA³⁰ (~92% methacrylation) from sodium hyaluronate (Lifecore), MeAlg (~70% modification) from sodium alginate (Sigma), and MeDex (~50% modification) from dextran (Sigma). MeMaHA with ~14% and ~10.5% methacrylate and maleimide modification, respectively, was synthesized via the coupling of the tetrabutylammonium salt of NaHA (HA-TBA) with 2-amino methacrylate hydrochloride (Sigma) and 2-amino maleimide trifluoroacetate salt (Sigma) (Supplementary Fig. S20). The chemical structures and ¹H NMR spectra of MeHA, MeAlg, MeDex and MeMaHA are provided in Supplementary Fig. S21, Supplementary Fig. S22, Supplementary Fig. S23, and Supplementary Fig. S24, respectively. The integrin binding peptide GCGYRGDSPG (Genscript; italics indicates cell adhesive domain) was conjugated to MeHA, MeAlg, and MeDex (754 µM, matching that used in the described physically crosslinked alginate studies), and to MeMaHA (1 mM) via 30 min reaction in pH 8.0 PBS at 25 °C prior to crosslinking. Passage 3 hMSCs (Lonza) were encapsulated either into MeMaHA (1 million hMSCs ml⁻¹) hydrogels using Michael addition reactions between MeMaHA maleimides and the MMP degradable peptide GCRDVPMS↓MRGGDRCG (Genscript; down arrow indicates cleavage site by MMP-2), or into MeHA, MeAlg, or MeDex (15 million hMSCs ml⁻¹) using photo-initiated free radical polymerization (Exfo Omnicure S1000 lamp with a 320–390 nm filter, exposure of 10 mW cm⁻² for 5 min) in the presence of 0.05 wt% Irgacure 2959 (I2959; Ciba), a photoinitiator chosen for its aqueous solubility and good cytocompatibility^{38 (link)}. For CD44 blocking studies, hMSCs were incubated with anti-CD44 (3/1000, mouse mAb CD44, Abcam) in a buffer (2 mM EDTA and 2% FBS in PBS) for 45 min on ice, washed twice in buffer, and resuspended in growth media prior to encapsulation. All gels were transferred to FBS-supplemented MEM-α (Invitrogen). MeMaHA hydrogels were secondarily photopolymerized at day 0 (“D0 UV”) or day 7 (“D7 UV”) by incubating with I2959 and exposing to UV light as described above. The elastic modulus of the hydrogels was measured via parallel plate compression testing at 10% ramped strain min⁻¹ as previously reported^{27 (link)}. For differentiation studies, following 7 days of incubation in growth media, hydrogels were transferred to a 1:1 mixture of adipogenic:osteogenic media (R&D Systems), with media changes every 3 days. For ROCK inhibition studies, selected −UV gels were treated with 10 µM Y-27632 (Sigma) daily during either the growth media (day 1–7) or mixed media (day 7–21) incubation periods.