Research on the preparation,biocompatibility and bioactivity of magnesium matrix hydroxyapatite composite material

2.2.1. Electrophoretic deposition experiment

Electrophoretic deposition method was used to prepare magnesium matrix hydroxyapatite composite material.

(1)

The preparation of the HA suspension. Scatter HA was added to the dispersion medium of anhydrous ethanol to prepare the suspension with concentration 0.02 kg/L. Little nitric acid (about 2% of suspending liquid volume) was added to the HA suspension as electrolyte. The pH value of suspension was adjusted to 4 ∼ 5 by aqueous ammonia. The suspension was shocked for 60 min by ultrasonic, boiled for 2 h by electromagnetic oven and aged for 10 days.

(2)

Pretreatment of magnesium alloy. AZ31B magnesium alloys were cut into two kinds of samples: 20 mm ∗ 2 mm ∗ 2 mm and 9 mm ∗ 9 mm ∗ 2 mm. All the magnesium alloy specimens were polished by SiC water proof abrasive papers with 320 ∼ 800 model, and then were cleaned for 10 min by ultrasonic in acetone, anhydrous ethanol and distilled water respectively. After cleaning, all the magnesium alloy specimens were dried by natural drying under low temperature.

(3)

Electrophoretic deposition. The mode of constant voltage 60 V was selected to deposit coating for 60 min magnesium alloy was used as cathode, lead sheet (20 mm ∗ 20 mm ∗ 2 mm) as anode. Two parallel pole plates with the distance of 10 mm were inserted into the HA suspension. Electrophoretic deposition device was shown in Fig. 1.

OPEN IN VIEWER

Fig. 1.

Electrophoretic deposition device.

(4)

Heat treatment. Relatively low temperature and high humidity environment was chosen to dry out the Mg/HA coating to minimize the crack. In order to make the coating densify, the Mg/HA coating was put in argon atmosphere of 400°C to preserve heat for 10 h.

(5)

Characterization of the Mg/HA coating. X-ray diffractometer was applied to analyze the phase composition of Mg/HA coating. Scanning electron microscope was used to observe the surface morphology of magnesium matrix hydroxyapatite biological composite material.

2.2.2. Animal experiment

The compatibility of magnesium matrix hydroxyapatite biological composite material was tested by animal implantation experiment.

(1)

Pretreatment of the implant. The implants were trimmed into cube specimen of 2 mm ∗ 2 mm ∗ 2 mm to implant into hind legs of the SD rats respectively. The phase composition of the coating was analyzed by X-ray diffractometer to verify the situation of HA coating and rule out other impurities (eliminate the interference of other foreign substance). Before the animal experiments, the implants were soaked in anhydrous ethanol solution about 30 min to prevent inflammatory reaction from bacterial infection.

(2)

Grouping and control design. The experiment was carried out in the animal experimental center of University of South China. Eight SD rats were selected with weight about 200 g, half males and half females. The SD rats were divided into two groups: (1) Implant group: the trimmed implants was implanted into left hind limb muscle of SD rats. (2) Control group: the SD rats were damaged in the left hind limb muscle by surgical knife without implant.

(3)

Surgery. After sterilizing the implants by anhydrous ethanol, 7% chloral hydrate as anesthetic was injected into SD rats with the dose of 0.005 ml/g. The SD rats were deeply anesthetized and fixed on operating table. The muscle tissue of left hind limb was selected as the location of implant, and depilated. The iodine complex disinfection solution was adapt in the conventional surgical disinfection and sterilization. The sterile operation towel was put under the experimental limbs of SD rats. The epidermis of experimental limb was sliced a cut about 1 ∼ 2 cm by surgical knife, then it would be implanted into muscle tissue. Finally, the SD rats were put in animal raising box without restricting the animal freedom after the wound was stitched up skillfully.

(4)

Histology detection. The experimental SD rats were fed for a period of time (2 weeks, 4 weeks and 8 weeks). Air embolisation method was used to kill the experimental SD rats after deeply anesthetized by 7% chloral hydrate. The implant surrounding tissue was cut from the experimental limbs immediately and put into plastic sample tube which filled tissue fixed solution, to be fixed. The implant surrounding tissue was taken to do tissue section and HE dyed, and to be observed by biomicroscope for organizational analysis.

2.2.3. SBF immersion test

SBF immersion test was used to inspect biological activity of magnesium matrix hydroxyapatite biological composite material. Magnesium matrix hydroxyapatite biological composite material prepared by electrophoretic deposition method was trimmed into experimental samples with the size of 9 mm ∗ 9 mm ∗ 2 mm. As a result of the electrophoretic deposition, the surface of experimental sample had coating on both sides. In order to avoid confusion, one side was used as experimental surface, and the other side whose coating was removed as the control surface. Before immersion test, the sample was soaked in anhydrous ethanol about 10 min, and then was cleaned twice in deionized water. After cleaning, the experimental sample was upright placed in plastic bottles which was filled with SBF solution (simulated body fluid). The scale between SBF solution volume and the plate area of sample is about 100 ml/cm². Then the experimental samples and plastic bottles of SBF solution were put in constant temperature box of 37 ± 1°C for 4 weeks. The experimental samples were taken out from plastic bottles to clean twice in deionized water, then to dry naturally for XRD analysis.