Design of Sequential Delivery System

For all in vivo testing a calcium phosphate layer was precipitated prior to a PLLys-PLGlut PEM application for delivery of BMP-2 only (Fig. 4.1A) or BMP-2and FGF-2 (Fig. 4.2B). The theoretical delivery profiles of the single- and sequential- delivery coatings are illustrated in Fig. 4.1C and D respectively. For the in vivo studies, two different coating architectures were used: amorphous calcium phosphate with 8 bilayers of PEM (CaP-PEM8), or nanocrystalline calcium phosphate with 30 bilayers of PEM (bCaP-PEM30).

4.2.2 Material Fabrication

4.2.2.a Preparation of CaP-PEM8 coated Healos®

Healos® (DePuy Synthes Spine, Raynham, MA) was trimmed to a thickness ~ 1mm, then scaffolds were cut out with a 3.5 mm biopsy punch (Integra Miltex, York, PA). Scaffolds were

human bone morphogenic protein 2 (BMP-2, R & D Systems, Minneapolis, MN) was applied to the Healos® scaffolds by adding 2 μl of 250 μg/ml stock made in PBS to each scaffold and allowing it to adsorbed for 1 h at room temperature.

After BMP-2 adsorption each scaffold was placed in a 1.5 ml micro-centrifuge tube and 1 ml of SBFx5 Solution A (prepared as described in Ch. 2) that is used to precipitate an amorphous CaP layer on the scaffold was added to each tube. Tubes were left open and covered in parafilm with 8-10 small holes poked through the parafilm. Tubes were placed in a 37°C oven. After ~24 h tubes were removed from the oven and sonicated (with scaffolds still in reacted SBFx5 Solution A) for ~ 10 sec to remove loosely bound CaP. Each scaffold was then placed in a 96- well plate and rinsed 3x with MilliQ water.

Each scaffold was then placed in a 1.5 ml centrifuge tube (EMD Millipore, Billerica, MA) with a 0.65 μm filter-membrane. The micro-centrifuge tubes are made up of two compartments separated by the membrane; this allows the sample to sit submerged in solution on top of the membrane until tubes are centrifuged, which then forces the solution through the scaffold and through the membrane, leaving the supernatant separated from the sample. 300 μl of 1 mg/ml poly-L-Glutamic acid (PLGlut, Sigma, St. Louis, MO) in saline was added to each tube and allowed to adsorb to the scaffold for 10 min. Note that the membrane is of sufficiently fine porosity that it does not allow the solution to go through it until the centrifugation step therefore the scaffold is completely immersed in the solution. After 10 min, tubes were centrifuged for ~10 sec at 1000 rpms and the supernatants contained below the membrane were discarded. Each scaffold was then rinsed 3x with saline with centrifugation after each rinse. Then 300 μl of 1 mg/ml poly-L-Lysine (PLLys, Sigma, St. Louis, MO) in saline was added to each tube and

allowed to adsorb for 10 min, then centrifuged and rinsed 3x with saline. This procedure was repeated until 8 bilayers of PEM were adsorbed (1 bilayer = PLGlut-PLLys).

After PEM8 application, recombinant human fibroblast growth factor 2 (FGF-2, R & D Systems, Minneapolis, MN) was adsorbed by adding 2 μl of various FGF-2 working stocks (prepared in saline) to each scaffold and allowed to bind for 1 h at room temperature. The following doses were tested: 0, 0.25, 5, 100, and 125 ng FGF-2. After FGF-2 adsorption, the scaffolds were rinsed 3x in saline then put in 300 μl DMEM high glucose, pyruvate medium, (No. 11995, Gibco BRL, Invitrogen) and stored in a 8-20°C refrigerator until ready for implantation. Prior to implantation the scaffolds were centrifuged for ~ 10 sec at 1000 rpms. This entire procedure is illustrated in Fig. 4.2.

4.2.2.b Preparation of bCaP-PEM30 coated Healos®

Healos® scaffolds were cut and sterilized as previously described. A 2 μg dose of recombinant human bone morphogenic protein 2 (BMP-2, R & D Systems, Minneapolis, MN) was applied to the Healos® scaffolds by adding 2 μl of 1 mg/ml stock made in PBS to each scaffold and allowing it to adsorbed for 1 h at room temperature.

For bCaP application, the two-step SBF method described in Ch. 2 was used to produce a nanocrystalline bCaP layer rather than just the amorphous layer previously described. During BMP-2 adsorption SBF Solution A was prepared as described in Ch. 2. Scaffolds were added to the beaker equipped with a stir bar. The beaker was covered with saran wrap with 20-30 holes punched into it and placed on a stir plate set to match the mixing during Solution A prep (~ 130 rpms) in a 37°C oven. After ~24 h the beaker was removed from the oven and sonicated (with scaffolds still in reacted Solution A) for ~ 10 sec. Prior to immersion in Solution B, the scaffolds

were gradually dehydrated by being passed through a graded series of increasing concentrations of ethyl alcohol.

Solution B was prepared as described in Ch. 2. Scaffolds were added to the beaker equipped with a stir bar. The beaker was covered with saran wrap with 20-30 holes punched into it and placed on a stir plate set to match the mixing during Solution B prep (~ 130 rpms) in a 50°C oven. After 7 or 24 h, the beaker was removed from the oven and sonicated (with scaffolds still in reacted Solution B) for ~ 10 sec. The scaffolds were gradually dehydrated by being passed through a graded series of increasing concentrations of ethyl alcohol.

After bCaP deposition, each scaffold was then placed in a 0.65 μm micro-centrifuge tube and 30 bilayers of PLGlut-PLLys were applied as previously described for PEM8. After PEM30 application, a 5 ng recombinant human fibroblast growth factor 2 (FGF-2, R & D Systems, Minneapolis, MN) dose was adsorbed and allowed to bind for 1 h at room temperature. After FGF-2 adsorption, the scaffolds were rinsed 3x in saline then put in 300 μl DMEM high glucose, pyruvate medium, (No. 11995, Gibco BRL, Invitrogen) and stored in a 8-20°C refrigerator until ready for implantation. Prior to implantation the scaffolds were centrifuged for ~ 10 sec at 1000 rpms. This entire procedure is illustrated in Fig. 4.3.

4.2.3 Characterization

4.2.3.a Scanning Electron Microscopy (SEM)

The microscopic morphology of the coated scaffolds was characterized using SEM (JSM - 5900LV, Jeol USA Inc. Peabody, MA). Coated scaffolds were also cut in half, allowing examination of the innermost fibers of the Healos® scaffold via SEM.