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Structural, mechanical and in vitro studies on pulsed laser deposition of hydroxyapatite on additive manufactured polyamide substrate

mode and the maximum scanning area was 5 × 5 µm solution. The colour developed is then determined in
and 10 × 10 µm. The X-ray diffraction (PANalytical an ELISA reader at 570 nm of UV-absorbance wave-
X-Pert Pro) measurement was performed to observe length.
the crystalline phases in the coated layer. The presence Data were analysed using the student t-test. Results
of functional groups was detected using a FTIR Spec- were considered statistically significant when the P
trometer (Josco Instruments, Japan). The hardness and value was less than 0.05 (P < 0.05). Results were dis-
Young’s modulus of HA layer was identified using played as the mean ± standard deviation.
nanoindentaion (CSM instruments, USA). According
to ASTM F1501-94, pull-out based adhesion strength 3. Results and Discussion
of the HA layer was observed using a DeFelsko pull- 3.1 SEM with EDX Analysis
off adhesion tester.
From Figure 1A, it was evident that the layer was
2.5 In Vitro Analysis composed of dense droplet-like spherical particles of

2.5.1 Cell Culturing non-uniform size. Some isolated particles were also
identified on the layer, and this was due to scattering
The cellular response of coating was assessed in terms or spurious melting of target material resulting in
of cell viability and proliferation to the surface. MG- globular particles present on the coating. At higher
63 osteosarcoma cell was used in this study and it is magnification a cluster (agglomeration) of nanoparti-
suitable for screening a large number of samples for cles (Figure 1B) can also be observed. At 135 mJ of
cytotoxic compound and also used in the rapid evalua- laser energy, particles were ablated explosively and
tion of the biomaterial surface qualities. Cells were there are possibilities to ablate bigger particles directly
2
grown in 75 cm flasks containing Dulbecco’s modi- from the target [49] and forming a layer with some iso-
fied Eagle’s medium (DMEM; Sigma). The media were lated pin holes and voids which in turn results in a
supplemented with 10% fetal bovine serum (FBS; In- heterogeneous rough surface. Few literatures demon-
vitrogen), 1.5 g/L sodium bicarbonate, 10,000 units/mL strated that having a rough surface will stimulate bet-
penicillin, 10 mg/mL streptomycin and 25 µg/mL ter osteointegration [50,51] .
amphotericin B. Cells were cultured as monolayers in Figure 2A shows a typical texture of deposited sur-
culture flasks at 37°C under a humidified atmosphere face. The layer of HA has randomly distributed glo-
of 5% CO 2. bular particles with a thickness of 1.6 µm and has a
2.5.2 Cell Seeding on Hydroxyapatite Coated growth rate of 53.34 nm/min (as per Equation 1).
Substrate Generally, at higher laser energy, the kinetic energy of
ablated atom increases and the number of surface nu-
The coated substrate was sterilised with 70% ethanol cleating atom also increases and hence it shows the
for 30 min and autoclaved for 30 min, followed by faster rate of deposition. Figures 1C and 2B shows the
drying at room temperature for 2 h. Cells were seeded EDX spectrum and EDX mapping of surface and cross
5
approximately 1 × 10 cells/sample. Cell proliferation section respectively. The spectrum reveals the pres-
and viability was assessed and observed at specific ence of calcium and phosphate at a ratio of 1.7 which
time periods at 1, 7 and 15 days respectively. is nearer to the ratio of standard HA. Moreover, the

2.5.3 Cell Viability dense distribution of such ions was observed in the
EDX mapping at the cross section.
The viability of cells was assessed by standard 3-(4,5- Layer Thickness (nm )
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Growth rate = Equation (1)
(MTT) assay. This assay is based on the reduction of Coating Duration (min)
soluble yellow tetrazolium salt to insoluble purple 3.2 AFM Analysis
formazan crystals by metabolically active cells. Only
live cells are able to take up the tetrazolium salt. The The 3D morphology of HA layer obtained by AFM is
enzyme (mitochondrial dehydrogenase) present in the shown in Figure 3. A rougher texture relating hetero-
mitochondria of the live cells converts internalised geneity can be observed in Figure 3A, due to a mixed
tetrazolium salt to formazan crystals, which are purple mode of particle size and agglomeration of larger par-
in colour. Then the cells were dissolved in DMSO ticles in a number of valleys and some sharp tiny

88 International Journal of Bioprinting (2016)–Volume 2, Issue 2