The
effect of strontium incorporated Zn-Ca-P coating on the biodegradability of
AZ31 alloy was evaluated. The Sr content and deposition time were optimized and
coated on AZ31 alloy by chemical conversion technique. The coating formed with
1.5 wt.% Sr and 20 min phosphating time at 50° C with pH 2.5 completely covers
the surface of the alloy. Sr doped coated sample showed evolved hydrogen volume
and pH value three times lower than Zn-Ca-P coatings which implied the
controlled degradation of the coating. On immersion in Simulated Body Fluid,
this surface exhibits high bioactivity with the deposition of calcium phosphate
phases with Ca/P ratio of 1.55 which is close to that of hydroxyapatite,
mineral component of bone. Cytotoxicity evaluation with L969 cells showed that
Sr doped coatings exhibited 72% cell viability on resorbable magnesium alloys.

Key
words: Magnesium, Zinc Calcium Phosphate,
biocompatible, corrosion, Strontium, cell viability.

 

 

 

 

1. Introduction

Magnesium
alloys have been evolving as a promising biodegradable material due to its favourable
mechanical properties, biocompatibility and lightweight 1. Particularly, the
elastic modulus of magnesium is closer to that of natural bone which avoids
stress shielding effect 2. Moreover, magnesium is an important element found
in human body and involved in body metabolic activities such as protein
synthesis, muscle contraction and relaxation, energy transport etc. 3, 4. Low
levels of magnesium lead to Alzheimer’s disease,
asthma, attention deficit hyperactivity disorder (ADHD) 5. Even though
magnesium alloys have many favourable properties, electrochemical potential of
magnesium is in the active region in the EMF series leading to rapid corrosion,
increase in local pH, hydrogen evolution, loss of their mechanical integrity
before bone healing process 6, 7. The necessary requirement of biodegradable
implants is that the degradation rate should be matched with the healing rate
of bone 8. For new bone formation, magnesium alloy should maintain its
mechanical integrity at least for 3 months. At present, magnesium alloys are
used for biodegradable implant applications such as screws, pins and
stents. 

Mainly
three approaches have been developed to control the degradation rate of
magnesium such as alloying, surface modification and coating 9. Coating is
one of the key solutions to overcome the corrosion rate of magnesium alloy in
chloride containing environment and provides some barrier effect between the
material and the environment. Various surface treatment techniques such as
physical vapor deposition, electrodeposition, anodization, chemical vapour
deposition, microarc oxidation are available to achieve desired coating 10.
Long treatment time, high cost, high temperature, complex procedures are the
limitations of these techniques. In contrast, conversion coating technique is a
simple technique and can produce a highly adherent coating 11. Particularly,
phosphate conversion coatings are regarded as suitable alternative compared to
other conversion coating techniques to improve surface properties due to low
toxicity 12.

Zinc phosphate, calcium phosphate
(CaP) and zinc calcium phosphate (Zn-Ca-P) coatings are attractive for
biomedical applications, as the elements present in the coatings are essential
for human health 13-17. However, to be best of available literature, no
attempt has been made to study the effect of ions in the Zn-Ca-P coating to
further improve the bioactivity and corrosion resistance.
Among various dopant ions, strontium is of our interest due to its biological
performance and bioactivity. Hence Sr doped HA, bioactive glass, bone cement
have been synthesized 18-21. Sr doped CaP coatings have been investigated as
an implant coating material 22. In view the advantages of both the coating
material and coating technique, we have attempted the feasibility of Sr doped
Zn-Ca-P coating by conversion coating method. By adding strontium precursor in
the phosphating bath, strontium incorporated PCC coatings can be developed.
Hence the present work deals with the optimization of strontium content in the
zinc calcium phosphate conversion coating on magnesium AZ31 alloy and its
evaluation for bioactivity, corrosion resistance and cytocompatibility.

2. Experimental Procedure

2.1. Substrate preparation

The
substrate used in the present study is AZ31 magnesium alloy which was purchased
from Exclusive Magnesium, Hyderabad, India. The chemical composition of the
substrate is 2.9% Al, 0.88% Zn, 0.001% Fe, 0.02% Mn, and balance magnesium. The
substrate was polished with SiC paper up to 1200 grit. The substrate was washed
with double distilled water and ultrasonically cleaned and degreased with
acetone.

Zn-Ca-P
and Sr doped Zn-Ca-P coatings were prepared on the surface of AZ31 alloy in the
phosphating bath. The phosphating temperature was maintained at 50° C. The pH of
the bath was adjusted to 2.5 with the help of phosphoric acid. The composition
of the phosphating bath is 10g/L diammonium hydrogen phosphate ((NH4)2HPO4),
7 g/L zinc nitrate (Zn (NO3)2), 3 g/L calcium nitrate
(Ca(NO3)2), 3 g/L sodium nitrate (NaNO2), 1g/L
sodium fluoride (NaF). Sr doped zinc calcium phosphate coating was deposited on
the substrate by varying the strontium content (precursor used is strontium
nitrate) as 0.5, 1 and 1.5 wt.% with optimized pH of 2.5 at 50° C (optimized
temperature) for various deposition times i.e. 5, 10, 15, 20 and 30 min. Zinc
calcium phosphate coating was also deposited for comparison.

2.2 Surface characterization

 Fourier Transform Infrared (FTIR) spectra of
Zn-Ca-P and Sr doped Zn-Ca-P coatings of various compositions were recorded on
an FTIR spectrometer in the range of 400-4000 cm-1 with a single
reflection ATR accessory (Perkin Elmer Spectrum two, USA). Chemical composition
and phases of the compounds were analyzed using X-Ray powder diffractometer
(XRD, D8 DISCOVER, Bruker, USA) using Cu k? radiation at 40kV and
30mA at a scan rate of 0.02°. Scanning Electron Microscopy with
Energy-Dispersive X-ray spectroscopy (SEM, FEI, QUANTA 200, NETHERLANDS) was
used to characterize the surface morphology and elemental composition of
Zn-Ca-P and Sr doped Zn-Ca-P coatings. Wettability of undoped and doped samples
was measured using contact angle instrument (Easy Drop KRUSS, Germany) and SBF
is used as contact liquid at different locations.

2.3 Adhesion characterization

Films
with good adhesion strength are considered as a protective overcoat and are very
vital for the protection against corrosion. Hence adhesion of the coating was
tested as per ASTM (American Standards for Testing and Materials) D3359-09
using tape adhesion test 23. 25 squares were developed by cross cutting the
coating in both directions using cross hatch cutter and adhesive tape was
applied on the cross-cut area and pulled rapidly. Percentage of the adhesion
remaining was calculated using the equation                  

Adhesion
remaining (AR) %= (n/25) × 100                  
————————– Eq.1

Evaluation
was made by substituting the number of peeled squares (n) in Eq.1 as per the
ASTM standard (0%-5B, <5%- 4B, 6-15%- 3B, 16-35%- 2B and 36-65%-1B). 2.4 In vitro degradation and mineralization In vitro studies were conducted by immersing the samples in Simulated Body Fluid (SBF). The sufficient numbers of samples with equal dimensions were prepared by coating process as explained in section 2.1. The procedure for the preparation of SBF was reported by Kokubo et al 24. The uncoated and coated samples were immersed in the SBF kept at 37° C for 15 days. Hydrogen evolution test (HET) was carried out to study the evolved hydrogen from corroding specimen and degradation of magnesium. The procedure for the HET experiment was in accordance with the earlier report 23, 25. The initial pH of SBF was maintained as 7.2 and pH were noted down at different time intervals. Corrosion current density obtained from an electrochemical method is easy and corrosion behavior can also be monitored with time. However, due to the special electrochemical behavior of magnesium called negative difference effect, the polarization curve will be distorted and the reaction mechanism around corrosion potential is different from that of tafel region. One hydrogen gas molecule is generated by dissolving one atom of magnesium and corrosion rate is directly reflected by hydrogen evolution rate. Hence corrosion rate from hydrogen evolution test is reliable. Corrosion rate of the samples was calculated using the following equation,                                                        Corrosion rate (CR) =     ------------------- Eq.2   Where CR is the corrosion rate in mg/cm2/h, 'A' is the initial surface area in cm2, 't' is the immersion time and ? is the density of the sample (1.74g/cm2).  ?g is the multiplication factor for calculating the amount of magnesium from the evolved hydrogen volume as 1mL of H2 is given by dissolving 0.001083 and this value is used for the calculation. 2.5 MTT Assay L969 cells (from NCCS, Pune) were cultured with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% 100× antibiotic antimycotic liquid and incubated in a CO2 incubator at 37° C. A cell suspension with DMEM containing 1×105 cells were seeded on the sample which was placed in the 9 well cell culture plate and MTT assay was conducted by incubating for 72 h at CO2 incubator. 200 µL of cell culture medium was added to each well. Then 20 µL MTT solution was added followed with incubation for 4 h at 37°C. The amount of reduced formazan product shows the number of viable cells. Plate reader was used for the quantification of formazan by absorbance at 570 nm. All the experiments were performed triplicates. 3. Results and Discussion 3.1 FTIR Analysis FTIR spectra of Zn-Ca-P and Sr doped Zn-Ca-P coated samples formed with different strontium nitrate contents are shown in Fig.1. The phosphate coating shows two strong absorption bands between 900 and 1150 cm-1 which is due to the stretching vibration of PO43- group. The characteristic bending vibrations of phosphate groups are formed at 550, 610 and 730 cm-1 26. The broad band around 3250 cm-1 and small band around 1680 cm-1 correspond to OH- vibration 27. Although the spectra look similar to each other, increase in the peak intensity of PO43- vibrations around 1026 cm-1 with increasing strontium content in ZCP coating has been observed. The high intense peak of PO43- vibrations has appeared for 1.5 wt.% Sr doped Zn-Ca-P coating. 3.2 XRD Analysis Fig.2 shows the XRD patterns of pure and Sr doped Zn-Ca-P coated samples with various Sr(NO3)2 contents. All the XRD pattern closely resemble with each other. The main diffraction peak of (3 2 1) of zinc calcium phosphate (JCPDS 98-000-427) and (2 4 1) of zinc phosphate (JCPDS 98-001-8145) and (0 4 0) of strontium phosphate (JCPDS 96-153-3308) are clearly shown in Fig.2. It is evident from the XRD analysis that the zinc calcium phosphate coating contains zinc phosphate and zinc calcium phosphate phases. In addition, strontium phosphate phase is also appeared in Sr doped Zn-Ca-P coated samples 28. 3.3 Coating mechanism Chemical etching of phosphoric acid with magnesium produces a chemically favourable surface for the formation of the subsequent coating. Hydrolyzation of phosphoric acid derives H+ ions which in turn react with magnesium ions. Remaining Mg2+ ions react with HPO42- and produce strong adhesive phosphate layer Mg3(PO4)2) on the substrate which makes the medium more alkaline. The alkalization of the solution facilitates the precipitation of insoluble phosphates such as Zn2Ca(PO4)2, Zn3(PO4)2 and Sr3(PO4)2. The coating methodology was schematically illustrated in Fig.3. The deposition process contains following steps a) dissolution of magnesium b) local increase in pH c) precipitation of magnesium phosphate d) formation of alkaline pH surrounding the implant e) precipitation of Zn2Ca(PO4)2, Zn3(PO4)2 and Sr3(PO4)2. From XRD results, Zn3(PO4)2 and Zn2Ca(PO4)2 formation in the undoped coating and additional Sr3(PO4)2 formation in the doped coating is confirmed. Temperature, pH, treatment time mainly contribute in the formation of compact two layers. 3.4 SEM Analysis Fig.4 shows the surface morphologies of phosphate coatings at different phosphating time. Fig. 4 (a, b, c, d and e) shows the surface morphology of the samples after immersion in the Zn-Ca-P phosphating bath for 5, 10, 15, 20 and 30 min respectively. Few white deposits were randomly deposited on the surface during the initial period. As the phosphating time was increased, the size of the deposits became larger and covered the surface. The maximum surface coverage was found at 20 min deposition time. Some cracks were found in the coating due to internal stresses and also due to the evolved hydrogen from the dissolution of the magnesium of the alloy matrix. Morphology of Zn-Ca-P coating was totally different from that of Sr-doped Zn-Ca-P coating which consists of needle like morphology. Fig.4 (f, g, h, i, and j) shows the surface morphology of Sr doped Zn-Ca-P coated samples. The randomly scattered particles formed on Sr doped Zn-Ca-P coating at initial deposition time could not effectively protect the surface from SBF. In contrast, the coating formed at 20 min deposition time covered the entire surface, though some defective regions were visible. Normally there was a generation of hydrogen from the corroding magnesium due to the micro galvanic corrosion between the particle and its ?-magnesium matrix 29. But small voids in the coating offers pathways for the hydrogen and thus damage in the coating is very much reduced. Moreover, Sr doped samples described herein controlled the magnesium degradation greatly than other conversion coated samples.  Maximum surface coverage and presence of bioactive strontium phosphate phase combinedly seems to influence in protecting the magnesium degradation in SBF. The maximum surface coverage also minimized the diffusion of SBF through the coating. Strontium phosphate present in the coating has a low solubility product value (1× 10-31), which dissolves very slowly when implanted in the human environment 30. When the deposition time was extended up to 30 min, some of the crystal deposits were dissolved in the phosphating bath and the surface was exposed again (Fig. (e) and (j)). Hence 20 min deposition time is fixed as optimum deposition time and Zn-Ca-P coating with varying Sr(NO3)2 content was studied and the morphology of the coatings are shown in Fig.5. Low strontium nitrate content in Zn-Ca-P coating did not cover the entire surface of the alloy. As the strontium nitrate content was increased to 1.5 wt.%, the surface was entirely covered with slab like crystals. Insert shows the compactly arranged slab like crystal morphology. The coating formed at 50°C was more compact compared to low phosphating temperature. If coating temperature is further increased, the coating dissolves in the acidic medium. Hence 20 min deposition time, 50° C temperature and 1.5 wt.% strontium content seems to be optimal parameters for the formation of a crack free coating. 3.5 Contact angle measurements The contact angle values of uncoated, Zn-Ca-P and Sr doped Zn-Ca-P coated substrates are shown in Fig.6. The contact angle of the uncoated alloy is found to be 89°, whereas, for Zn-Ca-P and Sr doped Zn-Ca-P coatings, the values are 97° and 112° respectively. High contact angle values suggest that the surface energy of coated surfaces is low resulting in the hydrophobic nature of the sample. Hence, when a drop is placed on the surface, it does not spread on the surface and exhibits corrosion resistant behavior. 3.6 In vitro biomineralization The SEM-EDX analysis of AZ31, Zn-Ca-P and Sr doped Zn-Ca-P coated samples during the immersion in SBF for 15 days are shown in Fig.7. The surface of the uncoated AZ31 alloy was severely cracked. The deposited calcium phosphate peeled off from the surface due to the continuous corrosion reaction and hence less amount of calcium depositions can be seen on AZ31 after 15 days of immersion (Fig.7c).  In case of Zn-Ca-P coating, stable deposits were observed on the surface and size of deposits were found to increase from 38 µm to 43 µm during the immersion time. However, Sr doped Zn-Ca-P coating exhibits the presence of deposits on the 5th day of immersion itself and the entire surface was covered with deposits of larger diameter (54 µm) (Fig.7i) at the final immersion period. The strontium present in the coating seems to assist in the deposition of more calcium from SBF and the calcium, in turn, withdraws more phosphate from the solution. Hence calcium phosphate with larger diameter was deposited in case of Sr doped Zn-Ca-P coated samples. EDX spectra of AZ31, Zn-Ca-P and Sr doped Zn-Ca-P, coated samples after 15 days of immersion are shown in Fig.7 (j, k, l). All the spectra confirm the presence of Mg, Al, Zn, Ca, and P elements. The Mg, Al, and Zn elements are from the substrate and Ca and P elements are from SBF. The Ca/P ratio calculated from EDX results for AZ31 was 0.67, while the ratio of Zn-Ca-P and Sr doped Zn-Ca-P coating were 1.26 and 1.55 respectively. The highest Ca/P ratio as close to that of hydroxyapatite (Ca/P = 1.67) obtained for Sr doped alloy indicated the improved biomineralization and hence protection from corrosion. 3.7 Adhesion characterization Adhesion of the coatings was tested by cross cut adhesion test. The coating was present in 25 squares (n=0) after the test for both coatings.  Hence the value was substituted in eq.1 and AR % calculated was found to be 0% and it falls under the highest grade 5B. Both the coatings seem to have good adhesion property without much delamination of the coating. 3.8 Hydrogen gas evolution The hydrogen evolution results of the uncoated, Zn-Ca-P and Sr doped Zn-Ca-P coated samples in SBF are shown in Fig.8. The total evolved volume of hydrogen gas of AZ31 is 4.5 mL. In contrast, hydrogen evolution rate of all the coated samples shows significantly lower values than AZ31 that could be tolerated by the human body. This may be due to the difficulty in charge transfer from solution to the alloy surface leading to good corrosion protection of the coated samples. The decrease in hydrogen evolution rates of Zn-Ca-P and Sr doped Zn-Ca-P coating may also due to high adhesion strength of the coating. Moreover, the deposited corrosion products from SBF also seem to help in delaying the degradation process. Particularly 1.5 wt.% Sr doped Zn-Ca-P coating has lowest hydrogen evolution due to the thick chemically stable and low soluble protective layer. 3.9 pH Measurements The pH variation of SBF solution during the immersion of uncoated and coated samples for 96 h are shown in Fig. 9. The pH values of SBF during the immersion of coated samples are lower than that of the uncoated alloys over the entire immersion time. The low pH increase of Zn-Ca-P coating than AZ31 is due to the presence of corrosion resistant and chemically stable zinc phosphate and zinc calcium phosphate in the coating. Sr doped Zn-Ca-P samples showed lowest pH increase. This may be due to the presence of strontium phosphate which has low solubility product value. Once it is coated on the surface, it will not reprecipitate from the surface and deposits in the phosphating bath. Also, strontium draws extra calcium and phosphate from the phosphating bath leading to the formation of the thick and compact coating. 4.0 Corrosion rate calculation from Hydrogen Evolution Test The corrosion rate of AZ31 by conventional electrochemical methods may not be reliable due to negative difference effect of magnesium and hence the corrosion rate was calculated from hydrogen evolution studies using the Eq.2 and it is shown in Fig 10. The corrosion rate of AZ31, Zn-Ca-P, and 1.5 wt. % Sr doped Zn-Ca-P coated sample at the end of 96 hr is 2.42, 0.84 and 0.67 mg/cm2/h respectively. It is remarkable that the corrosion rate of Sr doped Zn-Ca-P coated sample is one fourth of AZ31 during the initial immersion time. 4.1 MTT Assay The cell viability of L969 cells on AZ31, Zn-Ca-P, and Sr doped Zn-Ca-P coated samples by MTT assay are shown in Fig.11. All the samples were found to be nontoxic with 60% cell viability at the end of 72 h. More viable cells were present in Zn-Ca-P and Sr doped Zn-Ca-P coated samples than that of AZ31. Sr doped Zn-Ca-P coated sample showed the highest number of viable cells compared to other samples which may be due to the chemical stability of the coating. 5. Conclusions Optimal parameters such as pH, temperature, phosphating time and strontium content for the deposition of Sr doped Zn-Ca-P coating using chemical conversion technique was obtained. Sr doped Zn-Ca-P coated sample exhibits low pH, evolved hydrogen volume and corrosion rate. Strontium phosphate does not easily dissolve in the SBF solution due to its low solubility product value which provides more protection in the coating. This coating showed improved bioactivity as deposits having Ca/P ratio closer to hydroxyapatite due to the higher induction of calcium and phosphorous by strontium from SBF solution. Hence Sr doped Zn-Ca-P coated AZ31 alloy may be a suitable degradable implant for orthopedic applications. Acknowledgement One of the authors, Dr. P. Amaravathy thanks, Department of Science and Technology-Science and Engineering Research Board (DST-SERB), Government of India for providing fellowship under National Post-Doctoral Fellowship (NPDF) scheme.