University of Pennsylvania Department of Bioengineering Aims/Objective & Hypothesis: Effect of Calcium Content on Bone Fracture Energy Group Tuesday - A1 The primary objectives of this experiment are to determine the calcium content via chemical extraction of calcium from 10 chicken bone samples and investigate the relationship between the calcium concentration in the bone samples and the associated energy required to fracture the bones, which will be determined through the pendulum fracture technique. Hypothesis: There is a positive linear correlation between the amount of calcium present in the chicken bones and bone fracture strength

University of Pennsylvania Department of Bioengineering Methods, Protocol & Equipment: 1.Fracture bone using same methods used in the Impact Pendulum Testing Lab 1.Cut off section of fractured bone near point of fracture 2.Wash out bone section with distilled water in order to discard bone marrow 2.Perform surface decalcification technique 1.Prepare chelating agent using 31.2 g NaH 2 PO 4, 28.4 g Na 2 HPO 4, 140 g EDTA and 2 L distilled water 2.Cut bone section into thin, flat (~1 g) sections for rapid decalcification 3.Suspend thin sections in 50 mL of chelating agent for 15 minutes 3.Perform colorimetric analysis of 3.5 mL samples of the decalcified solution 1.Stain decalcified solution with calcium detection reagent 2.Place stained solution in cuvette and measure absorbance using a spectrophotometer at 550 nm 3.Compare absorbance value to calibration curves in the calcium detection reagent literature EDTA

University of Pennsylvania Department of Bioengineering Proposed Results/Findings/Deliverables: Using the chemical extraction of calcium and the subsequent determination of calcium concentration via colorimetric analysis, the calcium content in bone samples will be calculated. Due to the fact that calcium is present in significant amounts in bone, there presumably exists a positive linear relationship between calcium content and the strength of the bone (energy required to fracture the bone.)

University of Pennsylvania Department of Bioengineering Potential Pitfalls: Methods Pitfall What if the Ca 2+ content in the small bone fragments is too small for the colorimetric analysis to analyze? EDTA too slow for six hour lab. Chromogenic tetracarboxylic acid may be used instead (2 min rxn time) Will we be able to back calculate meaningful Ca 2+ measurements when using such small concentrations? May have to increase specimen size and/or increase sample size (n). Statistics Pitfall What if there is no significant difference between Ca 2+ content in the different chicken specimens? Observed in the cross-sectional areas and bone masses of pilot lab Perform completely different test: Find calibration curve of strong decalcifying agent on known calcium tablet over time (% Ca 2+ lost vs. time) Do pre-hoc test on bones and then fracture Determine effect of time in EDTA on fracture strength Reliable results

University of Pennsylvania Department of Bioengineering Materials and Budget & Justification: Necessary Materials:Price ($): 1kg Sodium Orthophosphate g O-cresolpthalein mg EDTA Surgical Blades (10) Blade Holders (4) mg Calcium Tablets (140) Chicken (2-8 pack) 5.00 Total: $ Materials Already Available: Pendulum Apparatus Spectrophotometer Glassware