HARD TISSUE SCAFFOLD USING FOAMING/FREEZE- DRY TECHNIQUE

FABRICATION MATERIALS:Chemicals

• 8% w/v Gelatin (from porcine skin)

• 10% w/v Calcium Phosphate relative to Gelatin

• N-hydroxysulfosuccinimide (NHS)

• 1-Ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrocholoride (EDC)

• 70% ethanol

• Hydrochloric acid (HCL)

• DI water

FABRICATION MATERIALS:Instruments/Consumables

• Mechanical mixer
• -80C freezer
• 4C refrigerator
• -55C Freeze-dryer
• Hot plate with thermometer • pH meter

• Aluminum foil • Scaffold molds • Scale
• Glassware

DAY 1: PREPARE MIXTURE

• Heat 25mL of DI water to 50C°

• Add 2g of gelatin

• Add 0.2g of Calcium Phosphate

• Use pH meter to ensure pH

  remains under 7

DAY 1: FOAMING

• Foam mixture with mechanical mixer at 1500 rpm for 5 minutes

• Transfer foam to preformed molds.

• Place the molds into -80C freezer and leave overnight to solidify.

DAY 2: FREEZE-DRY

• Remove samples from freezer

• Place aluminum foil over

  molds

• Use 18 1⁄2 gauge needle to

  create multiple holes in foil for venting

DAY 2: FREEZE-DRY (Freezer paramiters)

Place samples in freeze dryer and run at -55C and 1Pa for 48hrs.

DAY 2: CROSSLINKING

• Crosslink samples using 4 mg/mL EDC and 0.5 mg/mL NHS in 70% ethanol (v/v) at 5C for 1 hour.

• Thoroughly rinse the scaffold in DI water

• Place molds into -20C freezer and leave overnight

DAY 3: SECONDARY FREEZE-DRY

• Run frozen samples through a secondary freeze dry at -55C and 1 Pa overnight

• Oncecompleted,store scaffolds in 4C refrigerator until further use.