Bone Cements.
CaP cement bars were prepared by mixing a Norian SRS (Norian Corp., Cupertino, CA) calcium phos phate powder with a buffered sodium phosphate liquid by hand and allowing the cement to set in a 27.4 3 2.4 3 2.6-mm mold. The prepared cement bars were allowed to set for three days, removed from the molds, and sterilized overnight under ultraviolet light.

Dissolution Study.
The CaP bone-cement bars were divided into three groups, and the dimensions were measured precisely. Group I was immersed in a 1.0 mol/L Tris buffer with 80 mmol/L NaCl (pH 7.4 at 37°C). Group II was immersed in 5% fetal bovine serum in Tris buffer with 80 mmol/L NaCl (pH 7.4 at 37°C). Group III was immersed in a tissue fluid substitute containing 93.9 mmol/L NaCl, 1.24 mmol/L K2H PO4, 0.66 mmol/L KH2PO4, 0.94 mmol/L MgCl2, 1.48 mmol/L of CaCl2, and 18 mmol/L of KHCO3 (pH 7.4 at 37°C).13 It is imperative to test the pH of all solutions at 37°C. All cement bars were immersed in dissolution media in a 2.5:1 solution to cement surface area ratio. The study was performed in a sterile and humidified 95% air, 5% CO2 atmosphere at 37°C for 21 days. The buffer media was collected and replaced daily. The volume withdrawn and pH were recorded. Each collected solution was saved for subsequent analysis of phosphate and calcium released.

Measurement of Inorganic Phosphate.
The amount of phosphate released in solution each day was measured colorimetrically by using the reaction of ammonium molybdate and ascorbic acid with the inorganic phosphate to obtain a molybdenum blue complex. The reaction was done in a 96-well microtiter plate. Each sample was diluted ten-fold to make a 100 mL solution. Solution A was made by combining two parts double distill deionized water, one part 5.0 N sulfuric acid (Baker Analyzed, J.T. Baker, Phillipsburg, NJ), one part 0.01 mol/L ammonium molybdate tetrahydrate (Sigma Chemical Co., St. Louis, MO) in water, and one part 10% ascorbic acid (Sigma Chemical Co.). Solution A was made fresh for each assay. 100 mL of solution A were added to the 100-mL sample dilutions. The complex was allowed to form for 0.5 hours at room temperature. It was subsequently read at 630 nm on a Dynatech MR5000 microplate reader (Dynatech Laboratories, Chantilly, VA).14–16

Measurement of Calcium Ion.
Flame atomic absorption was used to measure calcium ion release from each CaP bone-cement bar. The blank was prepared by adding 90.0 mL of double-distill deionized water to 10.0 mL of 10X 1% LaCl3, 20% HNO3. The samples were prepared by combining 0.5 mL of sample from dissolution media with 1.5 mL of 0.1% LaCl3, 2% HNO3. Additional dilutions were made with 0.1% LaCl3, 2% HNO3. The samples were measured at 422.7 nm and energy of 49 using a PerkinElmer 3030 Atomic Absorption Spectrophotometer (PerkinElmer Analytical Instruments, Norwalk, CT).

Flexural Strength.
Bone cement bars were divided into six time-period groups: days 0, 4, 8, 11, 15, and 21. The cement bars were incubated in their respective solutions, either 1.0 mol/L Tris, 80 mmol/L NaCl buffer solution, 5% fetal bovine serum in 1.0 mol/L Tris, 80 mmol/L NaCl solution or tissue fluid substitute for their respective time period in a sterile and humidified 95% air, 5% CO2 atmosphere at 37°C in a 32-well plate. Cement samples were incubated at a 2.5:1 solution to cement surface area ratio. The incubation media were discarded and replenished daily. On completion of the incubation time period, samples were removed from their wells and placed into clean, dry wells and stored at room temperature until analysis. Non-incubated CaP cement bars were set aside at room temperature in a clean, dry 32-well plate for 21 days. Using an Instron (Instron Corporation, Canton, MA), 3-point bending was used to determine the flexural strength of the samples before and after immersion in solution. The cross-head speed used was 1 mm/minute.