Rgd functionalized alginate

RGD-functionalized alginate (FMC BioPolymer) was reconstituted in MEM alpha (Thermo Fisher Scientific) to create a 2% w/v solution, as described previously.^{19 (link)} Recombinant human bone morphogenetic protein 2 (BMP-2, Pfizer Inc.) was reconstituted in a solution of 0.1% rat serum albumin (Sigma–Aldrich) in 4 mM hydrochloric acid and mixed with the alginate solution to yield 2 or 5 μg BMP-2 per 150 μl of final solution. This alginate/BMP-2 solution was gelled with the addition of calcium sulfate (Sigma–Aldrich) at a 1:25 volume ratio. Hydrogels were prepared under sterile conditions inside a laminar flow hood and stored overnight at 4°C before use in surgery the next day.

A 2% hydrogel (w/v) of RGD functionalized alginate (FMC BioPolymer, Newark, DE) containing recombinant human bone morphogenetic protein-2 (rhBMP-2, Pfizer Inc., NY) was made as previously described [37 (link), 40 (link)]. BMP-2 solutions were formulated in 0.1% rat serum albumin (RSA, Sigma) in 4 mM HCl and added in appropriate volumes to yield 30 μg of BMP-2 per 150 μl hydrogel or media volume. Alginate hydrogels were stored at 4°C overnight before use. Sterile biopsy punches (4 mm) were used to create cylindrical constructs from a 10 mm thick sheet of sterile collagen sponge (ACS) (Kensey-Nash Corp., Exton, PA). The BMP-2 solution was added drop-wise to the scaffold during surgery and incubated at room temperature for 15 minutes prior to implantation. The therapeutics were delivered in perforated poly(ε-caprolactone) (PCL) nanofiber mesh cylinders (4.5 mm dia. and 12 mm length) placed in the bone defect region [37 (link), 40 (link)]. Alginate hydrogel was injected through the perforations in the mesh cylinder while collagen sponge was placed inside the mesh cylinder before loading the BMP-2 solution. Identically prepared delivery vehicles were used for in vitro and in vivo experiments.

Detailed scaffold production methods are described elsewhere [31 ,32 (link)]. Briefly, RGD-functionalized alginate (FMC BioPolymer) was reconstituted in α-MEM (Thermo Fisher Scientific) at 2% w/v. Recombinant human BMP-2 (Pfizer) was reconstituted in a 0.1% solution of rat serum albumin (Sigma-Aldrich) and 4 mM HCl and mixed with alginate, yielding 2 μg BMP-2 per 120 μL. The solution was ionically cross-linked with a 0.21 w/v CaSO₄ slurry mixed at a 1:25 volume ratio. Sheets of PCL nanofiber mesh were generated by electrospinning a 12% w/v solution of PCL in 90:10 (v/v) of hexafluoro-2-propanol:dimethylformamide from a syringe at a flow rate of 0.75 mL/h and a 15–20 kV potential onto a grounded collector plate 20–23 cm away. PCL sheets were laser cut into rectangles with a chessboard grid of 23 × 1 mm circular perforations, rolled and glued into 5 mm cylinders using ISO 10993 UV-curing adhesive (Dymax).