3.1. FESEM
Fig. 1 shows the representative microstructure of the sintered ATZ nanocomposite. Two different phases can be observed. The lightest one corresponds to the Ce-TZP matrix with a particle size of 400-500nm and the darkest phase corresponds to alumina with an average size of 250nm. As it can be observed alumina grains are homogeneously distributed and no pores are observed.
3.2. Surface roughness
Sandblasting is a commonly used surface treatment and involves impacting with hard particles at high velocities on a surface in order to erode it and leave a roughened surface with expected higher wettability. FESEM micrographs of the modified ATZ surfaces by sandblasting with white corundum and silicon carbide for 90s and 15s are shown in Figure 2A-C. Sandblasting with white corundum and SiC particles <90 microns (Fig. 2 A and C, respectively), revealed a regular and slightly waved structure. However, the surface of the sandblasted ATZ with SiC particles between 90 and 250µm (Fig.2B) showed more irregular structure with visibly larger voids and grooves, increasing the surface roughness according to the results shown in Table 2 for ATZ samples. The mean roughness index, Ra, in case of samples sandblasted with SiC between 90 and 250 microns was significantly higher than the other sandblasted samples. According to the Altbrektsson and Wennerberg classification [21], the samples sandblasted with white corundum present a “smooth” surface roughness (Ra < 0,5 µm), while the samples sandblasted with SiC show “minimally rough” and “moderately rough” surface roughness. Similar results has been found by Sato et al. [22] where sandblasting by SiC particles resulted in surface roughness values larger than those by alumina particles.
Raw materials
|
Ra < 0.5 µm
|
0.5 µm < Ra < 1.0 µm
|
1.0 µm < Ra < 2.0µm
|
Ra > 2.0 µm
|
W.C.< 90 µm
SiC 250-90µm
SiC < 90 µm
N. S
|
Smooth*
0.465 ± 0.06
0.031 ± 0.10
|
Minimally rough*
0.580 ± 0.08
|
Moderately rough*
1.358 ± 0.11
|
Rough*
|
Table 2: Roughness values of the ATZ nanocomposite surfaces after sandblasting with corundum and SiC for 90 and 15 seconds (mean ± SD in µm).N.S (No Sandblasting).
3.3. XRD diffraction
The volumetric fraction of the monoclinic phase was measured on: (1) as-sintered surfaces, (2) as-sintered surfaces after sandblasting process, (3) as-sintered surfaces after sandblasting and its ageing process. As it can be seen in Table 3, the materials do not present spontaneous phase transformation on their surface after sintering and any ageing process. However, sandblasting processes leads to the transformation, under tension, of a part of the tetragonal zirconia to monoclinic zirconia. According to the results shown in Table 3, the particle size and the kind of material used for the sandblasted process have an effect on the transformation phase of ATZ composite [23]. Increasing the particle size increases the erosion of material and the transformation surface layer. This transformation process is reversible and, in every case, a posterior thermal treatment at 1200ºC for 15 minutes succeeds in transforming the totality of the monoclinic zirconia back to its initial tetragonal state [24].
Treatment
|
Monoclinic phase content (Vm total)
|
ATZ as-sintered
|
3,80% ±3%
|
ATZ as-sintered and ageing during 5h
|
2,10% ±3%
|
ATZ as-sintered sandblasting surface by white corundum < 90 μm, 60 s
|
24,73% ±3%
|
ATZ as-sintered sandblasting surface by white corundum < 90 μm 60 s ageing during 5h
|
27,00% ±3%
|
ATZ as-sintered sandblasting surface by SiC 250-90 μm, 15 s.
|
48,70% ±3%
|
ATZ as-sintered sandblasting surface by SiC 250-90 μm, 15 s ageing during 5h
|
50,00% ±3%
|
ATZ as-sintered sandblasting surface by SiC <90 μm, 15s
|
31,70% ±3%
|
ATZ as-sintered sandblasting surface by SiC <90 μm, 15 s ageing during 5h
|
36,00% ±3%
|
Table 3: Volumetric fractions of monoclinic phase zirconia present on different sandblasted surfaces.
3.4. In vitro biological assays
ISO 10993-5 [19] states that a material is considered non-cytotoxic when cell viability is above 70%. The potential cytotoxicity on SaOs-2 and hADMSCs was assessed by the MTS assay and the NRU method using tissue culture polystyrene (TCPS) as the blank. According to the results shown in Fig. 3A-B all of the studied samples allowed for higher than 90% cell viability, therefore, none of the surface modification treatments can be considered cytotoxic.
Haemolysis is the alteration, dissolution or destruction of red blood cells that results in hemoglobin liberation into the surrounding medium. According to Stanley´s classification criteria, a material is considered non-hemolytic for hemolytic indexes <2 while it is considered slightly hemolytic and hemolytic for hemolytic index values of 2-5 and >5, respectively. Different factors such as surface roughness, surface energy and surface tension and surface wettability can have an influence on the blood compatibility and it is shown that surface modification has a great potential for improving the hemocompatibility of biomedical materials and devices [25]. In this case, the studied ATZ nanocomposite showed a hemolytic index close to 0 (0.1-0.2) for all the surface modifications tested, which was <1% indicating nonhemolytic material.
ALP levels increase when active bone formation (osseous differentiation) occurs, as it is a by-product of this process. According to the results shown in Fig. 4 A and B a correct osteoblast differentiation of hADMSCs has been taken placed since all of the differentiated cells used for gene expression studies were stained and, consequently, osteoblast differentiation confirmed.
Product identity was confirmed by electrophoresis on ethidium bromide-stained 2% agarose gels in 1X TBE buffer, which resulted in a single product of the desired length. In addition, an iCycler iQ melting curve analysis was performed, which rendered single product specific melting temperatures. No primer-dimers were generated during the 40 real-time PCR cycles conducted. All polymerase chain reaction efficiencies were above 90% and linearity was high, with correlation coefficients (R2) above 0.989.
To quantify gene expression, the relative standard method (relative fold changes) was used and expression levels were determined for the ceramics and the control group (NA sample) by normalizing results with respect to β-ACTIN. For hADMSCs, a discreet increase was noticed in the relative expression of four of the studied genes for samples A, B and C when compared with the control sample. In Fig. 5, these increases are plotted. In the problem group, the genes ΒGLAP, CASPASE 3, IBSP and SPARC were up-regulated 2.27-fold for sample A, 1.57-fold for sample C, 3.10-fold for sample A, and 2.27-fold for sample B, respectively, with respect to the basal levels recorded for the endogenous control (β-ACTIN). However, these increases were only significant (p<0.05) in the case of IBSP for sample A.
In the case of the SaOs cells, an even more discreet increase was observed in the relative expression of two of the studied genes for samples A, B and C when compared with the NA sample (control). These results are also shown in Fig. 5. The IBSP gene is 1.55-fold up-regulated for sample B. The COL1A1 gene is up-regulated 2.95-fold for sample A, 2.85-fold for sample B and 1.74-fold for sample C with respect to the basal levels recorded for the endogenous control. In this case, differences between control and problem groups were not significant.