Biomimetic Apatite Coating as an Alternative to Plasma-sprayed Hydroxyapatite Coating for Promotion of Bone Apposition-the Coating Formation and in Vitro Study

Introduction: The calcium phosphate ceramics, in particular hydroxyapatite ( HA), has shown to be capable of conducting bone formation and forming a chemical bond to bone. Because of this osteoconductivity, HA ceramic has been applied in joint reconstruction in the form of coating on joint implants such as hip stems by plasma spraying technique. Direct bone apposition has been repeatedly demonstrated on the plasma-sprayed HA coating and thus permits the fixation of the coated hip stems and cups in bone without the use of polymethylmethacrylate (PMMA) cement. Today, the cementless plasmasprayed-HA-coated joint implants have been widely used, especially in Europe and Japan. However the concerns about delamination of the plasmasprayed HA coating and its potential of generating ceramic particles have affected the acceptance of this coating by American surgeons. In the United States, the porous-coated joint implants such as bead-coated porous stems and acetabular cups are often used in favor of the plasma-sprayed HA coated implants. The porous structure added on implants permits the ingrowth of bone tissue leading to the anchorage of the implants known as biological fixation. Since Ti and Co-Cr alloys currently used in the manufacture of porous-coated implants are not naturally presented in the body, direct bone apposition on these materials has rarely been observed. Examination on retrieval of porous-coated implants has revealed a low percentage of available area occupied by ingrown bone in the porous layers, suggesting a need for improvement of direct bone apposition and ingrowth of bone in the porous layers. One goal of this study is to grow an apatite coating substantially equivalent to bone mineral on the surfaces of implants in order to promote direct bone apposition. The coating is intended to be formed at a physiologically related temperature by soaking an implant in a physiologically-related solution and is referred to as the biomimetic apatite coating. The biomimetic apatite coating is expected to be accepted by more orthopaedic surgeons in the world than the plasma-sprayed HA coating. This paper will focus on the formation of the biomimetic apatite coating and its interaction with biological solutions and human bone cells. Materials and Methods: The aqueous solution used in this study for growing biomimetic apatite coating includes all major inorganic ions present the body, namely Na, K, Mg, Ca, Cl, HPO4 , HCO3 and SO4. The solution was prepared by dissolving NaCl, KCl, K2HPO4, MgCl2, Na2SO4 and NaHCO3 in de-ionized H2O, with the concentrations of NaCl, KCl, NaHCO3 , MgCl2 and NaSO4 being at their corresponding levels in human blood plasma. The concentrations of Ca , and HPO4 2in the coating source solution range from 2.5 mM to 15 mM and from 1.0 mM to 10 mM respectively. The pH of the asprepared coating source solution was adjusted to neutral pH using tris(hydroxymethyl)aminomethane-hydrochloric acid. The solution was loaded in a glass reactor and titanium specimens were then soaked in the solution. The temperature of the solution was kept constant in the range of 3760 C using a circulating bath. The pH of the solution increases as HCO3 ions release from the solution in the form of CO2. As a result of this event, apatite coating was then grown on the surface of titanium specimen. After formatioon, the coating was analyzed by several techniques including diffuse reflectance Fourier transform infra-red spectroscopy (DR-FTIR), thin-film xray diffractometer (XRD) and scanning electron microscopy (SEM). The composition of the coating was analyzed using atomic abosorption spectrometer (AAS) and spectrophotometer. The biomimetic apatite-coated titanium specimens were soaked in pure bovine serum and osteocalcincontaining PBS for 24 hrs. The surfaces of the samples were then characterized by DR-FTIR and SEM to study the adsorption of biological molecules. Human bone cells were obtained from explants of trabecular bone of one 14-year-old patient. The fresh bone specimens were dissected and then sequentially digested with collagenase solution. The harvested cells received primary culture and were then frozen in liquid nitrogen and finally stored. The frozen bone cells thawed and were cultured in Dulbecco’s minimum essential medium containing 10% fetal bovine serum until confluence before they were inoculated on the biomimetic apatite coated Ti6Al4V disks in 12well plates. After culturing for 3 and 7 days, the bone cells were fixed, dehydrated and dried before their morphology was examined in a SEM. Results and Discussion: Figure 1 is the record of the pH change of the solution with time. The increase of pH at the beginning results from the release of HCO3 from the solution in the form of CO2 that can be expressed as the following: HCO3 = CO2 + OH. The drop of pH indicates the formation of the apatite coating on titanium speciemens because it requires the reaction: HPO4 2= PO4 + H. Figures 2 A-B are SEM photographs showing the morphology and the uniform coating about 10 micrometers thick covering the titanium specimen. The coating is composed of many numerous nano-crystals of apatite growing together and forming a unique nano-porous structure (Figure 2B). Unlike plasma-spraying technique, which is mainly a line-ofsight process, our process allows the formation of apatite coatings on all areas of the implants to create a three-dimensional coverage. As shown in Figure 2C, a Ti6Al4V bead about 150 micrometers in diameters is fully coated with an apatite film. It is impossible to achieve such full coverage by plasmaspraying technique. Characterization of the apatite coating by XRD, DRFTIR, AAS and spectrophotometer confirm that the biomimetic apatite is the nano-crystalline carbonated apatite and contains Mg and Na in its structure. Thus, the biomimetic apatite is substantially equivalent to bone mineral in composition and structure. As shown in Figure 3, the biomimetic apatite coating is much more similar to bone apatite than the plasma-sprayed HA coating that is the mixture of many calcium phosphate phases including hydroxyapatite.The study of the interaction between the biomimetic apatite coating and bovine serum and osteocalcin-containing PBS has demonstrated the adsorption of biological molecules on the coating. In particular, the biomimetic apatite coating is effective in absorbing osteocalcin dissolved in a PBS solution. SEM examination on the morphology of the human bone cells cultured on the biomimetic apatite coating for 3 and 7 days reveals that the cells appear to spread and adhere well on the coating. The bone cells had a stellate shape with numerous filamentous extensions. As indicated in Figure 4, the cell formed focal contacts with the coating. Summary:The biomimetic apatite coating can grow on joint implants at a low temperature (37-60 C) in a physiologically-related solution containing Ca , Mg , HPO4 , HCO3 ions as HCO3 ions release from the solution in the form of CO2. The formation of the apatite coating can occur on all areas of complex implants to create three-dimensional coverage. The biomimetic apatite coating shows a strong affinity to osteocalcin, and can absorb the biological molecules in biological fluids such as bovine serum. Human bone cells forms focal contacts when cultured on the biomimetic apatite coating.