Zinc temperature of galvanizing pot
Bone tissue is a complex composite material composed of mineralized collagen fibers with an assortment of other proteins and cells. The mineral phase is composed of calcium-deficient carbonated HA (Liu, Wu, & de Groot, 2010). Calcium phosphate (CaP) coatings have been widely applied to various implant materials, such as titanium and stainless steel, in order to improve their biocompatibility and to increase bone growth at the implantation site (Avila et al., 2009). In particular, biomimetically deposited coatings have been observed to have excellent biocompatibility and the ability to promote osseointegration (Avila et al., 2009; Liu, Wu, & de Groot, 2010). The biomimetic approach is a low-temperature solution deposition technique that involves immersing the substrate material in SBF.
A typical composition for SBF (Table 10.1) is based on the inorganic ions present in blood plasma. The exact composition is highly variable in the literature, where attempts to improve deposition rates, promote the deposition of specific CaP phases, and optimize biocompatibility have led to research with modified SBF solutions. The underlying theme for these coatings is that the inorganic ions present in the coating bath are also present in physiological solutions.
The crystallinity and composition of the deposited calcium phosphate coatings are similar to the mineral phase observed in natural bone tissue, resulting in improved bioactivity and good resorption characteristics.
The coating bath temperatures are low, allowing co-deposition of biological molecules such as proteins and growth factors that would decompose at higher temperatures. Furthermore, this low-temperature process does not alter the substrate significantly during coating.
The composition, pH, and temperature of the coating bath as well as the surface chemistry of the implant have both been shown to play a key role in the nucleation and growth of CaP coatings from SBF. The deposition rate and phase of CaP deposited are influenced by numerous factors such as the Ca/P ratio, concentration of inorganic ions, pH, the presence of crystal growth inhibitors such as Mg2+, surface pretreatment, and coating post-treatments (Barrere, van Blitterswijk, de Groot, & Layrolle, 2002a,b; Feng et al., 2000; Hu et al., 2006; Kuroda, Ichino, Okido, & Takai, 2002; Liu, et al., 2002).
Calcium phosphate coatings have been reviewed as a means of controlling both the degradation rate and the biocompatibility of magnesium alloy implant materials (Shadanbaz & Dias, 2012). The deposition of CaP on magnesium alloys is both helped and hindered by the nature of the substrate itself. A general mechanism for biomineralization is outlined below (Gray-Munro & Strong, 2009):
Step1:Mg(s)→Mg2+(aq)+2e−and2H2O(l)+2e−→H2(g)↑+2OH−(aq)
Step2:H2PO4−(aq)→HPO42−(aq)→PO43−(aq)
Step3:(10−x)Ca2+(aq)+xMg2+(aq)+6PO43−(aq)+2OH−(aq)→(Ca(10−x)Mgx)(PO4)6(OH)2(s)
Upon immersion in an aqueous CaP solution, anodic dissolution of magnesium accompanied by cathodic reduction of water readily occurs (Step 1). The corrosion of the substrate surface gives an increase in magnesium ion concentration and pH near the solution/metal interface. The increase in pH results in a shift in the phosphate ion equilibrium toward deprotonated species (Step 2). This increase in pH further affects the solubility of calcium phosphate, resulting in heterogeneous nucleation and growth of calcium-deficient, magnesium-rich HA at the interface (Step 3).
It has been well documented that magnesium ions can readily substitute into the crystal lattice of HA. The biomimetic process is typically done under ambient conditions; therefore, carbonate ions are also introduced into the crystal structure of the deposited CaP. As the pH of the bulk solution increases, homogeneous nucleation and growth of calcium phosphate crystals in solution can also occur, resulting in the deposition of additional CaP crystals at the surface. Due to its high reactivity, the magnesium alloy substrate is an active participant in the coating deposition mechanism, resulting in faster deposition rates and no need for complex surface modification strategies to induce CaP nucleation at the surface. The disadvantage is that the CaP layers produced at the interface are essentially a corrosion product; therefore, they tend to be amorphous, non-uniform, poorly adhered layers. This has a detrimental impact on their corrosion resistance. Moreover, the composition of this initially deposited inner CaP layer is often the same regardless of coating bath composition. This is illustrated in Figures 10.7 and 10.8. Figure 10.7 is an SEM image of a biomimetic CaP coating deposited on the AZ31 magnesium alloy. These figures are results from experiments conducted in our research laboratory. The coating bath was a mixture of 3 mM CaCl2 and 1.8 mM Na2HPO4 to give a Ca/P ratio of 1.67/1, the stoichiometric ratio of HA. Two distinct layers were observed. The inner layer is composed of a uniform layer of agglomerated CaP particles. The observed cracks are most likely due to dehydration of the inner layer. The outer layer is composed of larger agglomerates of spherical particles that deposit on the surface after formation in the bulk solution. Infrared microscopy confirmed that both the inner layer and outer layer are HA minerals.
Figure 10.7. SEM image and ATR-FTIR spectra of calcium phosphate coating on Mg AZ31 deposited from a 3 mM CaCl2/1.8 mM Na2HPO4 solution.
Figure 10.8. SEM image and ATR-FTIR spectra of calcium phosphate coating on Mg AZ31 deposited from a 2 mM CaHPO4 solution (pH adjusted to 5 with H3PO4).
Figure 10.8 is an SEM image of a biomimetic calcium coating deposited from a chloride-free solution of CaHPO4. It can again be readily observed from these images that two distinct layers are formed. The inner layer is identical in morphology and chemistry to that observed from the CaCl2/Na2HPO4 coating bath, indicating that the nature of this layer is mainly controlled by dissolution of the substrate itself. However, the outer layer is composed of plate-like crystals of brushite. The deposition of brushite crystals rather than apatite in this instance is likely due to the decreased degradation rate of the magnesium alloy surface in the absence of chloride ions. This results in a slower increase in pH of the bulk solution and homogeneous precipitation of brushite crystals in the coating bath.
This type of duplex coating structure—consisting of a thin, compact inner layer and a porous crystalline outer layer—has also been reported in the literature for biomimetic calcium phosphate coatings on magnesium and its alloys (Yang, Cui, & Lee, 2011).
The study of biomimetic CaP coatings on magnesium alloys has been the subject of several research articles in the last decade. The exact surface pretreatment, coating bath composition, and coating deposition conditions vary widely. In particular, the coating times range from several hours to several days. However, there are some common themes:
The deposition temperature is typically 37 °C.
The initial coating bath pH is typically between 7 and 7.4. A couple of articles reported the use of lower pH values, from 5 to 7. Notably, one paper reported that CaP coatings deposited from a pH 6 coating bath are more uniform, less porous, and have improved corrosion resistance over those deposited at physiological pH (Lu, Chen, Huang, & Yan, 2012).
The coating bath composition varies, but for the most part it consists of either SBF (Table 10.1) or modified simulated body fluid (m-SBF), which contains higher concentrations of Ca2+ and HPO42−.
In the absence of surface pretreatment, CaP is readily deposited on magnesium and its alloys via the previously presented mechanism. It has been observed that the number of biomimetic layers deposited (Zhang, Zhang, & Wei, 2008) has a significant impact on the degradation rate of the coated material. The degradation rate of a poorly crystalline HA coating deposited from m-SBF decreased significantly when a second layer was deposited on top of the first. Coating bath chemistry can also affect the nature of the calcium phosphate phases formed. In a coating bath where CaCl2 was used as the calcium source, a dicalcium phosphate dihydrate coating was reported (Yanovska et al., 2012), whereas a Ca(NO3)2-containing solution resulted in the deposition of HA (Yang et al., 2009; Yanovska et al., 2012). The crystallinity of the deposited coatings was also shown to increase in the presence of a magnetic field (Yanovska et al., 2012). Finally, when pure magnesium, the AZ21 alloy, and a 0.5 wt% Ca magnesium alloy were exposed to bone cells in cell culture medium for 18 days, a bone-like matrix was deposited on the surface of these materials (Pietak, Mahoney, Dias, & Staiger, 2008). Although the concept of depositing coatings using bone cells has not been widely explored due to the risk of immogenicity (Rahmany & Van Dyke, 2013), it could be a promising method for optimizing the biocompatibility and degradation rate of these materials.
A variety of surface pretreatments have been explored as a means for improving the adhesion and corrosion resistance of these biomimetically deposited CaP coatings. Some general pretreatment strategies that have been reported on magnesium alloys include polishing, acid etching, pre-calcification, anodizing, heat treatments, hydrothermal treatment, and MAO.
It has been reported that surface roughness has an impact on the corrosion resistance of CaP coatings. Good corrosion resistance was observed for polished samples, whereas the decrease in degradation rate was not as significant for roughened samples (Nguyen, Waterman, Staiger, & Woodfield, 2012). A CaP coating deposited from m-SBF for 5 days on a polished magnesium alloy AZ91 surface was found to consist of amorphous, calcium-deficient, carbonate-substituted HA. The coating was found to decrease the dissolution rate of the magnesium alloy by a factor of 5. The coating also enhanced cell adhesion in the short term (Lorenz et al., 2009). Likewise, biomimetic CaP coatings with some corrosion resistance were deposited on rough acid etched samples from both a simple calcium phosphate solution (Gray-Munro & Strong, 2009) and m-SBF (Lu et al., 2012). These coatings were both observed to consist of amorphous, calcium-deficient, carbonate-substituted hydroxyapatite.
Anodizing, MAO, and heat treatment have also been proposed as pretreatments (Cortes, Lopez, & Mantovani, 2007; Hiromoto et al., 2008; Liu, Hu, Ding, & Wang, 2011). In each case, calcium phosphate coatings were deposited on the pretreated surface upon immersion in SBF or m-SBF solutions, and improved corrosion resistance was observed.
Hydrothermal treatment of pure magnesium by immersion in 100 °C water for various times was also investigated to increase the number of hydroxyl groups at the surface (Waterman et al., 2011). Surface modification of titanium implants with hydroxyl groups is essential for the nucleation of CaP, although this is not critical for CaP nucleation on magnesium. However, hydrothermal pretreatment did impart improved corrosion resistance to the CaP-coated magnesium in SBF in comparison to non-pretreated samples.
The most popular pretreatment strategy reported thus far is precalcification of the surface prior to biomimetic CaP coating. Precalcification involves deposition of a calcium-rich layer via a chemical method. In several cases, a prelayer of CaHPO4·2H2O (brushite) was deposited prior to immersion in SBF or m-SBF (Hu et al., 2010; Shadanbaz et al., 2013; Xu et al., 2012a,b). Precalcification was shown to increase the deposition rate of biomimetic calcium phosphate coatings. Furthermore, the existing brushite precalcification layer was converted to hydroxyapatite during final coating formation. Precalcification by deposition of CaHPO4 (monetite) was also reported (Shadanbaz et al., 2013). The monetite pretreatment was shown to have better corrosion resistance than brushite in several different types of cell culture media. This may be due to its decreased solubility in aqueous solution. Finally, precalcification by deposition of a Ca(OH)2 underlayer has been shown to produce biomimetic CaP coatings with fewer defects, better corrosion resistance, and self-healing capability (Waterman et al., 2012).
Post-treatment via sealing of biomimetic CaP coatings has also been attempted (Keim, Brunner, Fabry, & Virtanen, 2011). Four different post-treatments were applied to SBF-formed layers:
soaking in 1 M NaOH at room temperature for 24 h,
heat treatment at 150 °C in air for 1 h,
heat treatment in steam for 1 h, and
soaking in boiling water for 1 h.
Only soaking in NaOH and heating in steam resulted in noticeable sealing of cracks and pores in the CaP coating. Furthermore, post-treatment with these methods did not improve the corrosion resistance of the magnesium substrate. Interestingly, the authors determined that deposition of CaP coatings from Dulbecco's Modified Eagle cell culture medium resulted in more protective CaP coatings with increased biocompatibility. The exact mechanism of this increased corrosion resistance was not reported; however, the composition of the cell culture medium is significantly different than traditional SBF solutions because it is rich in amino acids and sugars.
Biomimetically deposited calcium phosphate coatings have been shown to significantly improve both the corrosion resistance and biocompatibility of a variety of different magnesium alloys. Further research is needed to optimize the corrosion resistance, learn to control the phase of CaP deposited, and enhance long-term cell adhesion and proliferation.