Acidic gelatin

Using established methods, GMPs were fabricated using acidic gelatin (Nitta Gelatin, Osaka, Japan) with an isoelectric point (IEP) of 5.0 and crosslinked in 40 mM glutaraldehyde (Sigma-Aldrich, St. Louis, MO), followed by glycine quenching of unreacted glutaraldehyde [16 (link), 37 (link)]. Following lyophilization, GMPs of 50–100 μm in diameter were obtained through sieving and EO sterilized for 12 hours prior to GF loading. GMPs were then partially swelled with either phosphate buffered saline (PBS) or a GF solution at a ratio of 5 μL solution to 1 mg dried GMP for 15 hrs at 4°C before hydrogel encapsulation according to previously described methods [15 (link), 17 (link)]. In particular, 225 μL of PBS, IGF-1 solution, or BMP-2 solution was combined with 45 mg of GMPs. Recombinant human IGF-1 (Peprotech, Rocky Hill, NJ) was reconstituted with PBS to a GF loading concentration of 7.95 μg per mL solution and loaded onto GMPs to give a total of 5.6 ng IGF-1 per hydrogel construct. Recombinant human BMP-2 (Peprotech, Rocky Hill, NJ) was reconstituted with a mixture of ddH₂O and PBS to a GF loading concentration of 712 μg per mL solution to give a total of 1 μg BMP-2 per hydrogel construct.

NiPAAm, dimethyl-γ-butyrolactone acrylate (DBA), glycidyl methacrylate (GMA), acrylic acid (AA), 2,2’-azobis(2-methylpropionitrile) (azobisisobutyronitrile, AIBN), N,N’-methylenebisacrylamide (MBA), piperazine (PiP), glycine, and glutaraldehyde were purchased from Sigma Aldrich (Sigma, St. Louis, MO) and used as received. Anhydrous 1,4-dioxane, diethyl ether, and acetone in analytical grade; and water, acetonitrile, chloroform, and methanol in HPLC-grade were purchased from VWR (Radnor, PA) and used as received. Phosphate buffered saline (PBS) (powder, pH 7.4) was obtained from Gibco Life, Grand Island, NY. Ultrapure water was obtained from a Millipore Super-Q water system (Millipore, Billerica, MA). Acidic gelatin (isoelectric point = 5.0) was obtained from Nitta Gelatin (Osaka, Japan). Complete osteogenic medium (COM) was made from minimal essential medium α modification (αMEM) (Gibco Life, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Cambrex BioScience, Walkersville, MD), 10⁻⁸ M dexamethasone, 10 mM β-glycerol 2-phosphate, 50 mg/L ascorbic acid, and 10 mL/L antibiotic-antimycotic solution (Gibco, Life, Grand Island, NY). Live/Dead viability/cytotoxicity kit was purchased from Molecular Probes (Eugene, OR). The calcium assay was purchased from Genzyme Diagnostics (Cambridge, MA).