1. Alginate Solution Preparation

1. Dissolve 200 mg of sodium alginate (non-irradiated) or 400 mg of 8 MRad irradiated sodium alginate in 10 ml double distilled water and shake for 1 hr for non-radiated alginate and 15 min for irradiated alginate. Store the alginate solution at 4 °C overnight. Filter the alginate solution with a sterile 0.2 µm syringe filter before alginate microbead fabrication.

2. Alginate Microbead Fabrication

1. Disinfect the electrostatic bead generator and syringe pump with 70% ethanol and place them in a Class II Biological Safety Cabinet (Figure 1).
2. Place a glass basin with a magnetic stir bar inside the bead generator.
3. Set the arm electrode of the bead generator 9 cm above the basin.
4. Connect the electrode cable of the bead generator to the knurled screw 2 of the arm electrode, and pour 80 ml of SrCl₂ solution into the basin.
5. Load 5 ml of 0.2 µm filtered alginate solution into the syringe and rubber tube. After connecting the rubber tube to the arm electrode, switch on the syringe pump at 5 ml/hr for 2 min to expel the air inside the tubing and deliver the alginate solution to the tip of the nozzle. Turn off the syringe pump.
6. Next, switch on the encapsulator and then, the syringe pump to commence microbead generation. Set the alginate flow rate at 5 ml/hr and the voltage at 5.8 kV on the encapsulator. Discard the microbeads generated during the first two minutes (or the initial 0.5 ml alginate solution pumped out of the syringe), as these microbeads tend to be irregularly sized and shaped.
7. Collect subsequent microbeads in 0.2 M strontium chloride solution. Turn off both the syringe pump and the encapsulator (in that order) after pumping the pre-planned volume of alginate solution. Repeat this for subsequent batches of microbead fabrication. Upon completion, turn off the syringe pump first, followed by the encapsulator.
8. Store the microbeads in 20 ml of 0.2 M strontium chloride solution at 4 °C overnight to complete cross linking and stabilize the gel.

3. Size Measurement of Alginate Microbeads

1. Collect 0.5 ml of alginate microbeads with a plastic pipette and place it on a glass slide. View the microbeads under an optical microscope at 10X magnification. Take ten images of the microbeads with a microscope CCD camera. Save the images with scale bar (500 µm) in TIFF format at resolution 2,048 x 1,536.
2. Using the length tools in ImageJ, measure the size of the microbeads and scale bar (Figure 2). Convert the microbeads length from pixel to micrometer.
   1. Click on the line tools and draw a line across the middle of the alginate bead.
   2. Click "Analyze" on the menu bar and select "Measure". A pop-up window will appear.
   3. Repeat steps 3.2.1-3.2.2 to measure all alginate beads within the image. Measure the scale bar on the image.
   4. Convert the diameter of the alginate bead to the actual length by using the formula:length of alginate bead/length of scale bar x 500 µm. For example, 1.420 (diameter measured by ImageJ) / 2.657 (scale bar length measured by ImageJ) * 500 µm = 267 µm.
3. Consider the average size of 100 microbeads (mean ± standard deviation) as the representative size of each batch of microbeads.

4. Sterilization

1. Collect the microbeads using a 100 µm nylon strainer and wash the beads with double distilled water.
2. Using a spatula, transfer all the microbeads made from 0.1 ml alginate solution into a 2 ml microcentrifuge tube and cover with gauze to prevent drying.
3. Finally, sterilize the microbeads by autoclaving using liquid mode (115 °C, 15 min) or in accordance with manufacturer's specifications. Add 1.5 L of distilled water to the chamber to prevent beads from drying.

5. Protamine and Heparin Coating

1. Inside the BSL-2 hood, incubate the sterile microbeads with 1 ml of 2 mg/ml protamine solution (sterilized using a 0.2 µm syringe filter) for 1 hr at room temperature.
2. After incubating the microbeads for 1 hr (Step 5.1), collect 150 µl of the protamine solution for the micro bicinchoninic acid (microBCA) test (section 6).
3. Wash the protamine coated microbeads twice with double distilled water. Spin down using a bench top centrifuge at 200 x g for 3 min at room temperature. After centrifugation, aspirate the water using a syringe.
4. Incubate protamine coated microbeads with 1 ml of 0.5 mg/ml heparin solution (sterilized using a 0.2 µm syringe filter) for 30 min to create polyelectrolyte complex (PEC).
5. After incubating the protamine coated microbeads for 30 min (Step 5.4), collect 400 µl of the heparin solution to determine heparin content (section 7).
6. After incubation, wash off the unbound heparin from the PECs by washing twice with double distilled water.

6. Protamine Content

1. Perform the microBCA test according to manufacturer instructions. Briefly, add 150 µl protamine solution (collected before and after incubation with microbeads) into a 96 well plate. Add 150 µl microBCA working solution.
2. Use albumin solution (0, 0.5, 1, 2, 5, 10, 20, 40 and 200 µg/ml) as calibration standards.
3. Incubate the mixture for 60 min at 60 °C. Measure the absorbance with a spectrophotometer at 562 nm.
4. Use the standard curve to determine the protamine concentration of each unknown sample according to manufacturer's instructions.
5. Determine the protamine content of the microbeads by subtracting the total amount of protamine in the coating solution (before incubation with microbeads) from the amount of protamine remaining in the coating solution (after incubation with microbeads).

7. Heparin Content

1. Prepare the 10 ml of working solution by dissolving 4 mg toluidine blue and 20 mg sodium chloride in 0.01 N hydrochloric acid.
2. Add 400 µl of sample (from step 5.5) to the working solution at a ratio of 2:3 and vortex for 30 sec.
3. Add 600 µl of n-hexane (equivalent volume to the working reagent solution) and vortex the mixture to extract the toluidine blue heparin complex.
4. Aspirate 200 µl of the aqueous phase by syringe after phase separation.
5. Measure the amount of un-extracted toluidine blue contained in the aqueous phase using a spectrophotometer at 631 nm.
6. Prepare heparin standard solutions of 0-20 µg/ml.
7. Plot the 631 nm reading of each heparin standard vs. heparin concentration in µg/ml. Use the standard curve to determine the heparin concentration of each sample.

8. Confocal Image of Layer-by-layer Structure

1. Fabricate protamine, heparin and NELL-1/BMP-2 fluorescent analog CF-405 protamine (blue), CF 594 heparin (red) and FITC labeled NELL-1/FITC labeled (green) BMP-2 + heparin + protamine according to manufacturer's technical datasheet.
2. Coat 100 µg microbeads with 300 µl of fluorescent analog (coating method as described in 5.3-5.6) CF-405 protamine (blue) (2 mg/ml, 1 hr incubation), CF 594 heparin (red) (0.5 mg/ml, 30 min) and FITC labeled NELL-1/FITC labeled (green) BMP-2 (1.5 mg/ml, overnight). Wash the microbeads twice with distilled water to eliminate the unbound fluorescent protamine, heparin and NELL-1/BMP-2.
3. Observe the layer-by-layer structure (Figure 3) by using a confocal microscope at 10X magnification.⁷

9. BMP-2 and NELL-1 Uptake and Release

1. Load 13.3 µl of 1.5 mg/ml of BMP-2 or NELL-1 solution on 100 µg of PEC. Incubate PEC at 4 °C under 30 rpm shaking for 10 hr.
2. Immerse the microbeads in 1 ml of phosphate buffered saline (PBS) at 37 °C with constant shaking (30 rpm).
3. Collect 1 ml of the supernatant and replace it with 1 ml PBS after 1, 3, 6, 10 and 14 days.
4. Evaluate the uptake and release efficiency of BMP-2 using the ELISA method according to the manufacturer's protocol. Evaluate the uptake and release efficiency of NELL-1 using the carboxybenzoyl quinoline-2-carboxaldehyde (CBQCA) protein assay method according to the manufacturer's protocol.
5. Determine cumulative release at time (t):
   Cumulative release at time (t) = Release at time (t) + Previous release at time (t-1).
6. Plot cumulative release of BMP-2 and NELL-1 against time.

10. In Vitro Bioactivity of NELL-1

Note: The bioactivity of NELL-1 released from PEC was assessed by measuring its ability to increase the expression of alkaline phosphatase (ALP) in rabbit bone marrow stem cells (rBMSC).

1. Seed 20,000 rBMSCs per well in a 24-well plate and allow them to grow for one day with 1 ml of Dulbecco's Modified Eagle's Medium (DMEM) + 10% Fetal bovine serum (FBS) at 37 °C and 5% CO₂.
2. After 24 hr, replace the medium with 1 ml of an osteogenic medium (DMEM supplemented with 10% FBS, 2% penicillin streptomycin, 50 µg/ml ascorbic acid, 10 mmol/L beta-glycerophosphate, and 10^-8 mol/L dexamethasone) for 7 days at 37 °C and 5% CO₂.
3. Place 300 µg PEC-NELL-1 (from step 8.2) and PEC inside cell culture inserts (TC insert) to keep the PECs separate from the cells (this avoids the washout of PEC microbeads during the osteogenic medium change). Place TC insert into the 24 well plate for 14 days.
4. Once every three days, aspirate 1 ml of the osteogenic medium by placing a needle outside the TC insert, and replace with 1 ml of fresh osteogenic medium.
5. After 7 and 14 days of incubation, determine ALP activity with an ALP assay kit in accordance with the kit manufacturer's protocol.
   1. Lyse cells with assay buffer containing 0.1% TritonX-100 at 4 °C for 10 min. Scrape off adhered cells using a cell scraper. Incubate cell suspension at 4 °C under agitation for at least 60 min.
   2. Centrifuge the cell suspension at 2,500 x g at 4 °C for 10 min. Collect the supernatant for the ALP assay.
   3. Add 50 µl of serially diluted alkaline phosphatase standard solution from 200 to 0 ng/ml to the wells of a 96 well plate. The final amounts of alkaline phosphatase standard are 10, 5, 2.5, 1.2, 0.6, 0.3, 0.15, and 0 ng/well.
   4. Add the supernatant from step 10.5.2 (50 µl/well) and dilute with dilution buffer.
   5. Add 50 µl of p-nitrophenyl phosphate (pNPP) substrate solution into each well. Mix the reagents by gently shaking the plate for 30 sec. Incubate the mixture for 30 min in the dark. Measure the absorbance at 405 nm by plate reader.
   6. Calculate ALP activity using the calibration curve.
6. Determine protein content using the microBCA protein assay kit according to manufacturer's instructions. Normalize the ALP activity by dividing ALP activity by protein content.

11. Cell Viability

1. Incubate 200 mg of PEC-NELL-1 with 1 ml of DMEM +10% FBS at 37 °C for 24 hr for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
2. Seed 2,000 rBMSCs per well (in 100 µl of DMEM with 10% FBS) in a 96 well plate and incubate for 1 day at 37 °C, 5% CO₂.
3. Replace DMEM + 10% FBS with 100 µl of PEC-NELL-1/PEC extract and incubate at 37 °C, 5% CO₂.
4. After 1 day or 3 days of incubation, add 10 µl of 5 mg/ml MTT solution and further incubate at 37 °C, 5% CO₂ for 4 hr in darkness.
5. Add 100 µl DMSO solution to each well to dissolve formazan crystals.
6. Determine the absorbance at 570 nm using a microplate reader.
7. Calculate the relative growth rate:
   

12. Packaging into Scaffold and BMP-2 & NELL-1 Loading

1. Pack the PECs into the pores of a bioresorbable medical grade Polycaprolactone – tri-calcium phosphate (mPCL-TCP) scaffold using a sterilized spatula inside a BSL-2 chamber.
2. Add 1.5 mg/ml solution of BMP-2 or NELL-1 onto the mPCL-TCP scaffold packed with PEC and incubate overnight at 4 °C.