SA Bender (BA), Dental student, University of Texas Health Science Center at San Antonio, School of Dentistry, San Antonio, TX, USA.
JP Schmitz (DDS, PhD), Associate Professor, Department of Oral and Maxillofacial Surgery, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
JL Ong (PhD), Associate Professor, Department of Restorative Dentistry, Division of Biomaterials, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

Calcium phosphate (CaP) cements are used as implant materials for facial bone augmentation as well as cranioplasty and skeletal augmentation. They are used to improve alignment, fixation, and stabilization of healing fractured bones. Other uses of CaP bone cements have included their potential in delivering high concentrations of antibiotics and hormones to fractured bone sites. Success of CaP cement relies on the osteoconductive effect it has on adjacent bone by its inherent degradative property and its eventual replacement by normal bone. The setting reaction involving equimolar quantities of tetracalcium phosphate (Ca4(PO4)2) and dicalcium phosphate anhydrous (CaHPO4) with water proceeds by hydrolysis, dissolution, and precipitation of hydroxyapatite [Ca10(PO4)6(OH)2] from supersaturated solution. Acidified (H3PO4) and basic (CaOH2) byproducts are neutralized so that the setting reaction occurs close to a pH of 7 and is complete at 37°C in four hours. Because the reaction occurs at a nearconstant rate as determined by titration, inference is that zero-order reaction kinetics prevail.

Like the hydroxyapatite and CaP coatings on dental and orthopedic implants, it is also known that the initial cellular response to bone cement is partly dependent on the proteins adsorbed onto the surfaces. It has been hypothesized that as proteins from the biological fluids come in contact with synthetic surfaces, cellular adhesion, differentiation, and the production of the extracellular matrix production will be affected. Previous studies suggested that the CaP bone cement implant replacement by bone was postulated to occur through a combination of implant resorption coupled with osteoconduction. In other studies, no resorption of the CaP cement implant was reported regardless of the occurrence of fibrous union. Because dissolution of CaP from the surface of the implant in the human body contributes to the bioactivity of the CaP surface, the dissolution property of CaP bone cement may be critical to implant success. Therefore, the objective of this study was to simulate clinical conditions by evaluating the dissolution of CaP bone cement in different biofluids. The effect of dissolution on the CaP bone cement’s flexural strength was also investigated.

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.

Calcium Release.
An increasing amount of Ca21 released from bone cements was observed after incubating the bone cements in all solutions throughout the 21-day study period (Figure 1). There was no statistically significant difference (analysis of variance, P . 0.05) in Ca21 dissolution from cements immersed in 1.0 mol/L Tris buffer solution as compared with both 5% fetal bovine serum solution and tissue fluid substitute.

Inorganic Phosphate Dissolution.
Similar to the calcium release, an increasing amount of P51 dissolution was also observed throughout the 21-day study period (Fig. 2). There was no statistically significant difference in the dissolution properties of P51 between the 1.0 mol/L of Tris buffer (0.370 6 0.02 mg/mm2) and 5% fetal bovine serum solutions (0.247 6 0.06 mg/mm).2 However, for cements that were incubated in tissue fluid substitute, the dissolution of P51 was significantly less (0.03 6 0.002 mg/mm)2 throughout the study period as compared to the cements that were immersed in 1.0 mol/L Tris buffer and 5% fetal bovine serum solution.

Flexural Strength.
There was no significant difference in strength measurements of bars immersed in all media throughout the 21-day period (Fig. 3). However, regardless of solution, the strength increased to an average of 7.78 6 1.82 N in comparison with 3.19 6 0.93 N for non-immersed samples after 21 days.

Fig. 1. Release of Ca ions from bone cements after 21 days in different media.
Solid line: Tris buffer; Broken line: tissue fluid substitute; Dotted line: Tris buffer and fetal bovine serum.



Fig. 2. Release of P ions from bone cements after 21 days in different media.
Solid line: Tris buffer; Broken line: tissue fluid substitute, Dotted line: Tris buffer and fetal bovine serum.



Fig. 3. Flexural strength of the bone cements before and after immersion in different media for 21 days.

Discussion.
Acidic, basic, and buffered physiological salt solutions have been used as electrolytes for evaluating the dissolution of CaP implant materials. Furthermore, in recent studies, the success of CaP implants was suggested to be dependent on their ability to resorb or degrade; thereby allowing cellular penetration. However, irrespective of the degree of crystallinity, all CaP implants will degrade or dissolve to some degree. Because the amorphous phases are expected to dissolve first, it is possible that they control the initial biological response at the ceramic surface.
Protein content of the microenvironment has also been shown to affect dissolution properties of both hydroxyapatite and CaP test samples. It has been speculated that the anionic nature of proteins causes the attraction of Ca ions. However, the influence of proteins did not have a significant effect on the dissolution quantities of calcium in comparison with media without proteins. This could be the result of the fact that calcium dissolution has been shown to be strongly dependent on the microenvironment of the surrounding media. Therefore, because the media was removed and replaced daily, it was never allowed to become supersaturated with calcium in any of the three media tested. For this reason, the solutions in which the CaP cement bars were immersed were always undersaturated with respect to Ca, subsequently causing the release of Ca from the bars into the microenvironment.

References.

Kurashina K, Kurita H, Kotani A, et al. Experimental cranioplasty   and skeletal augmentation using an alpha-tricalcium phosphate/dicalcium phosphate   dibasic/ tetracalcium phosphate monoxide cement: A preliminary short-term experiment   in rabbits. Biomaterials. 1998;19: 701-706.
Shindo ML, Costantino PD, Friedman CD, et al. Facial Skeletal augmentation   using hydroxyapatite cement. Arch Otolaryngol Head Neck Surg. 1993;119:   185-190.
Goodman SB, Bauer TW, Carter D, et al. Norian SRS Cement augmentation in   hip fracture treatment. Clin Orthop. 1998;348:42-50.
Otsuka M, Matsuda Y, Kokubo T, et al. Drug release from a novel selfsetting   bioactive glass bone cement containing cephalexin and its physicochemical propoerties.   J Biomed Mater Res. 1995;29:33-38.
Hamanishi C, Kitamoto K, Tanaka S, et al. A self-setting TTCP-DCPD apatite   cement for release of vancomycin. J Biomed Mater Res. 1996;33:139-143.
Otsuka M, Yoneoka K, Matsuda Y, et al. Oestradiol release from self-setting   apatitic bone cement responsive to plasma-calcium level in ovariectomized rats,   and its physicochemical mechanism. J Pharm Pharmacol. 1997;49:1182-1188.
Bohner M, Lemaitre J, Van Landuyt P, et al. Gentamicin-loaded hydraulic calcium   phosphate bone cement as antibiotic delivery system. J Pharm Sci. 1997;   86:565-572.
Kveton JF, Friedman CD, Piepmeier JM, et al. Reconstruction of suboccipital   craniectomy defects with hydroxyapatite cement: A preliminary report. Laryngoscope.   1995;105:156-159.
Gresser JD, Hsu SH, Nagaoka H, et al. Analysis of a vinyl pyrrolidone/poly   (propylene fumarate) resorbable bone cement. J Biomed Mater Res. 1995;29:   1241-1247.
Chow LC. Development of selfsetting calcium phosphate cements. J Ceram   Soc Jpn. 1991;99:954-964.
B. Kasemo and J. Lausmaa. The biomaterial-tissue interface and its analogues   in surface science and technology. In: Davies JE ed. The Bone- Biomaterial   Interface. Toronto: University of Toronto Press; 1991:19-32.
Costantino PD, Friedman CD, Jones K, et al. Hydroxyapatite cement. I. Basic   chemistry and histologic properties. Arch Otolaryngol Head Neck Surg. 1991;   117:379-384.
Majeska RJ, Wuthier RE. Studies of matrix vesicles isolated from chick epiphyseal   cartilage. Association of pyrophosphatase and ATPase activities with alkaline   phosphatase. Biochem Biophys Acta. 1975;391:51-60.
Chen P, Toribara T, Warner H. Microdetermination of phosphorus. Anal   Chem. 1956;28:1756-1758.
Heinonen J, Lahti R. A new and convenient colorimetric determination of inorganic   orthophosphate and its application to the assay of inorganic pyrophosphatase.   Anal Biochem. 1981;113:313- 317.
Hergenrother P, Martin S. Determination of the kinetic parameters for phospholipase   C (Bacillus cereus) on different assay based on the quantitation of inorganic   phosphate. Analytical Biochemistry. 1997;251:45-49.
Hurson S, Lacefield W, Lucas L, et al. Effect of the crystallinity of plasma   sprayed HA coatings on dissolution. In: Transactions of the 19th Annual   Meeting of the Society for Biomaterials. Minneapolis, MN: Society for Biomaterials;   1993: 223.
Driessens FCM, van Dijk JWE, Borggreven JMPM. Biological calcium phosphates   and their role in the physiological of bone and dental tissue. I. Composition   and solubility of calcium phosphates. Calcif Tissue Res. 1978;26:127- 137.
Ducheyne P, Kim CS, and Pollack SR. The effect of phase differences on the   time-dependent variation of the zeta potential of hydroxyapatite. J Biomed   Mater Res. 1992;26:147-168.
LeGeros RZ, Orly I, Gregoire M, et al. Substrate surface dissolution and   interfacial biological mineralization. In: Davies JE, ed. The Bone-Biomaterial   Interface. Toronto: University of Toronto Press; 1991:76-88.
Whitehead RY, Lucas LC, Lacefield WR. The effect of dissolution on plasma   sprayed hydroxylapatite coatings on titanium. Clin Mater. 1993;12:31-39.
Tofe AJ, Watson BA, Cheung HS. Dense HA and microporous HA resorption. In:   Transactions of the 19th Annual Meeting of the Society for Biomaterials. Minneapolis,   MN: Society for Biomaterials; 1993:338.
Maxian SH, Liesch JB, Tiku ML, et al. Evaluation of hydroxyapatite coatings   of varying crystallinity: Cell culture and non-interference fit surgical studies.   In: Transactions of the 19th Annual Meeting of the Society for Biomaterials.   Minneapolis, MN: Society for Biomaterials; 1993:336.
Radin S, Ducheyne P, Berthold P, et al. Effect of serum proteins and osteoblasts   on the surface transformation of a calcium phosphate coating: A physicochemical   and ultrastructural study. J Biomed Mater Res. 1998;39:234-243.
Weng J, Liu Q, Wolke JGC, et al. Formation and characteristics of the apatite   layer on plasma-sprayed hydroxyapatite coatings in simulated body fluid. Biomaterials.   1997;15:1027-1035.
SA Bender, JD Bumgardner, MD Roach, et al. Effect of protein on the dissolution   of HA coatings. Biomaterials. 2000;21:299-305.
Daculsi G. Biphasic calcium phosphate concept applied to artificial bone,   implant coating and injectable bone substitute. Biomaterials. 1998;19:1473-1478.