THE ANALYSIS OF BOVINE SERUM ALBUMIN BINDING AFFINITY FOR XENOGRAFT COMPARED TO A SYNTHETIC PARTICULATE BONE GRAFT MATERIAL.

Abstract

WORKING HYPOTHESIS: Binding of albumin to various bone graft materials is correlated with the surface porosity of these materials, and therefore the binding of albumin to xenograft is stronger than its binding to synthetic bone graft. NULL HYPOTHESIS: There is no significant difference in the binding strength of albumin to xenograft than its binding to synthetic bone graft materials. Objectives: The increased availability of commercially produced bone graft materials versus autogenous bone makes these materials a desirable product in guided bone and tissue regeneration procedures. The use of commercially produced bone graft materials also provides the opportunity of the addition of certain biologic materials in order to enhance the healing response and to overcome the predominantly inactive nature of most graft materials on the market. The development of an adequate carrier of biologic agents is a crucial step in the creation of a bioactive graft material. This study uses an easily manipulated model protein to study specific characteristics of protein binding and release on two different bone graft substrates commonly used as calcified scaffolds in guided bone and tissue regeneration. This experiment was completed as a first phase in the establishment of a protocol for the future investigation of other relevant proteins that may be important in bone and tissue regeneration. Methods: Bovine serum albumin (BSA) dissolved in physiologic buffered saline solution was poured over 100 mg of either xenograft or synthetic particulate grafting material, and incubated for 24 hrs. at 4°C. The quantity of BSA protein adsorption to the grafting material surface was determined by removing all liquid from the wells after the 24 hr. incubation period, followed by quantification of protein concentrations using the bicinchoninic acid (BCA) protein assay reagent kit. In order to analyze the kinetics of protein release, 1 ml of phosphate-buffered saline (PBS) wash was added to all wells, stirred and removed from each well. This was followed by the addition of 1 ml PBS to all wells and removal of 1 ml of liquid at intervals of 1, 3, and 7 days. Protein concentrations were quantified using the BCA protein assay, and the results were analyzed using a two-way ANOVA. Scanning electron microscopy was performed on samples of xenograft and synthetic graft particles prior to BSA exposure, as well as at days 1 and 7 following the initial 24 hr. incubation and the subsequent PBS wash. Energy-dispersive X-ray spectroscopy was also used to analyze the elemental components of the xenograft and synthetic graft material after BSA treatment. Results: Scanning electron microscopy revealed a more porous surface texture and collagenous appearance of the xenograft graft material, versus the synthetic graft. The energy-dispersive X-ray spectroscopy showed a noteworthy difference between the elemental composition of the xenograft and synthetic graft material. A lower concentration of protein was shown in solution after the initial 24 hr. incubation period in the xenograft samples possibly indicating that more protein was bound to the xenograft particles than the synthetic bone. The remaining solution from the xenograft samples throughout the kinetics of release analysis showed more albumin protein released over time as compared to the synthetic graft samples. Conclusions: This study revealed that xenograft material showed a more porous surface structure and greater binding affinity for bovine serum albumin as compared to the synthetic material. The protocol described in this study is a useful model system for future studies to investigate other proteins involved in wound healing, bone remodeling, and angiogenesis. Protein binding and kinetics of release should be explored on alternative mineralized scaffolds or carrier systems in order to determine an adequate delivery mechanism that allows for sustained release during the optimum time frame for modulation of the healing process. Future experiments should focus on identification of an ideal transport medium for bioactive agents that will direct cells into the osteogenic process to restore new bone and periodontal supporting tissues. The engineering of a material that has the quality of extended release of proteins necessary for the healing cascade has the potential to unlock the key to periodontal regeneration.