Surface Structures And Properties Of Calcium Phosphate Bioceramics And The Relation With The Special Adsorption Of Proteins

Protein adsorption on biomaterials is the initial event after implantation, and the adsorbed protein plays an important role in the subsequent cellular function and determines the success of the implant. Having the similar inorganic compositions with human bone tissue, calcium phosphates have been widely used as bone repair or substitution materials in clinic, and their excellent biological properties have also been adequately confirmed. So it is one of the basic subjects for us to investigate the adsorption behavior of proteins, especially some bone-related proteins on calcium phosphates, as is helpful to explore the nature of the bioactivity and osteoinduction of calcium phosphates.Because the current studies on protein adsorption mainly concentrate on single protein adsorption on hydroxyapatite, and the mechanism of protein adsorption on various calcium phosphates and the relations between the structure and chemical properties of calcium phosphates and the competitive adsorption behavior of various proteins on them were not well known. In this paper, various protein systems including on single protein, binary protein, serum and in vivo diffuse chamber were chosen, and the behavior of protein adsorption in vitro & in vivo on calcium phosphate ceramics with different structure and phasic compositions were investigated.It is known that the behavior of protein adsorption on biomaterials be determined by the properties of biomaterials and the proteins. According to the measurements of contact angles and zeta potentials, the average contact angles of HA and BCP ceramics were 71.7±0.8 and 77.4±1.3°, and the zeta potentials of them were negative in pH7.4 PBS. Based on the adsorption experiments, BSA adsorption on calcium phosphate ceramic particles was rapid and could be described by the First-Order Equation. The isotherms of BSA adsorption on them could be well fitted using the Langmuir model. XPS analysis showed that either the surface Ca or P atoms of HA and BCP, its binding energy shifted to some extent, proving that the chemical bonds around Ca or P atoms on the surface had altered after BSA adsorption. The alterations of the chemical bonds around Ca or P after BSA adsorption imply that the electrostatic interaction might be a negligible factor for BSA adsorption on calcium phosphate ceramics.Comparing with the theoretical values, the saturated amounts of BSA adsorption on calcium phosphate ceramic particles were lower, resulting in the lower surface coverage of BSA on them. BCP has lower surface net charge than HA and the hydrophobic degree of BCP is slightly stronger than that of HA, these may be the reasons why BCP absorb more BSA molecules than HA in PBS. One hand, BCP particles had fewer negative zeta potential and surface net charge than HA, thus the BSA molecules are easier to reach the surface of BCP particles due to the lower repulsive forces between BCP particles and BSA molecules. On the other hand, BCP shows the higher contact angle and formed the stronger hydrophobic bonding with BSA molecule than HA, so there are more BSA molecules accumulating on the surface of BCP particles and consequently the amount of absorbed BSA become increased.From the zeta potential measurements and BSA adsorption on calcium phosphate ceramic particles at various conditions, it could be found that the alteration of pH, ionic strength, Ca²⁺ and PO₄^3- concentration strongly affected the polarity and magnitude of the zeta potential of calcium phosphate ceramics and the amounts of the adsorbed BSA. The coherence between the variation of BSA adsorption, and that of the zeta potentials proved that the electrostatic interaction plays an important role in protein adsorption on calcium phosphate ceramics.According to the competitive adsorption of BSA and LSZ on calcium phosphate ceramic particles, the similar competitive adsorption behavior of BSA and LSZ on various calcium phosphates was found. The differences of the adsorbed amounts and adsorption rates between BSA and LSZ confirmed the competitive nature of protein adsorption on calcium phosphates. LSZ had higher affinity for them than BSA and would preferentially bind to the surface. The mechanism could also be explained by the electrostatic interaction, together with the structure stability of protein molecules. The mechanism of competitive adsorption of BSA and LSZ was different from their concentrations in binary BSA/LSZ solution. When the concentrations of BSA and LSZ were equal, LSZ with smaller size would dominate in the initial adsorption on the surface. However, with the increase in BSA concentration and the decrease in LSZ concentration, BSA would dominate in the initial adsorption on the surface. Because of the surface coverage of BSA on calcium phosphates was lower, so there were a number of unoccupied reactive points on the surface. Thus, LSZ with smaller size and positive net charge would reach to those unoccupied reactive points because of the electrostatic attractions between LSZ and BSA. At higher bulk concentrations, less unfolding of BSA occurs due to the interactions between BSA molecules, resulting in the declined interaction between BSA and calcium phosphate ceramic particles. As a result, the previously adsorbed BSA could be gradually replaced by LSZ with higher affinity for calcium phosphate ceramics. It is known that the bone growth factors, such as BMP-2, TGF-β, and IGF etc., are basic proteins and have lower molecular weight. As thus, the experimental results also imply that those above growth factors with the similar properties as LSZ would concentrate on the surface when calcium phosphate implants are exposed to body fluids, as is undoubtedly important for the osteoinduction of calcium phosphate ceramics.Based on the protein adsorption on calcium phosphate ceramics with different structure under rat serum incubation and in vivo diffuse chamber implantation conditions, the porous structure of calcium phosphates was favorable for protein adsorption and its contribution to the amount of the adsorbed protein was higher than that of the specific surface area of calcium phosphates. In vitro serum incubation, the behavior of protein adsorption on calcium phosphate ceramics was not single layer adsorption but multi-layer one.The SDS-PAGE, Western Blotting and ELISA analyses for the adsorbed proteins showed that calcium phosphate ceramics not only adsorb those abundant proteins in blood and tissue fluids but also adsorb some bone-related proteins including on fibronectin and TGF-β1. Specially, the amounts of the adsorbed TGF-β1 increased with the increase in the incubation time under in vivo diffuse chamber implantation conditions, proving that calcium phosphate ceramics could concentrate on the bone growth factors such as TGF-β1 and So on. HA, BCP andβ-TCP showed different ability to adsorb TGF-β1, and the relative amount of the adsorbed TGF-β1 onβ-TCP was lower than that of HA and BCR Besides, the porous BCP adsorbed more TGF-β1 than HA andβ-TCP, as could be one of the reasons that BCP has the best osteoinduction.