**3. Zirconia-toughened alumina (ZTA) and alumina-toughened zirconia (AZT)**

#### **3.1. Background**

The demand of structural ceramics has led to an increased interest in Alumina–Zirconia composites for biomedical [21, 61] and dental implant application [62, 63]. Two composite materials can be prepared: ZrO2 reinforced with alumina particles, which is denominated alumina toughened zirconia (ATZ), and Al2O3 reinforced with zirconia particles, which is known as zirconia-toughened alumina (ZTA). Thus, higher fracture values can be reached if compared with the monophase ceramics [64] (**Table 2**).


**Table 2.** Physical and mechanical properties of ATZ and ZTA data are extracted from Faga et al. [85].

groups. Akagwa et al. [52] reported a similar bone to implant interlock in loaded and unloaded zirconia implants, but a crestal bone loss higher around the former group. In favor of the clinical use of Zirconia, it must be cited its maintenance of bending strength of over 700 MPa after immersion in 95°C saline solution for over 3 years [53]. Furthermore, zirconia blanks did not show any significant mechanical detriment even after being embedded in the medullary cavity

Scarce are the clinical studies dedicated to the long-term performance of zirconia implants. The short follow-up period and the often small sample size hinder their quality of evidence, so that Andreiotelli and coauthors [29] could only include three retrospective cohort studies on one-piece zirconia dental implants in their systematic review, reaching in total 231 patients and 416 implants. The studies by Mellinghoff et al. [54] and Oliva et al. [55] investigated, respectively, 189 and 100 zirconia implants and estimated 1-year survival rates of 93 and 98%. Almost all of the failures occurred during the healing phase, as only one implant failed after prosthetic reconstruction due to fracture. Lambrich and Iglhaut [56] observed 127 zirconia and 234 titanium implants for a mean period of 21.4 months. Notably, in this study, the survival rate of zirconia implants was similar to that of titanium in the mandible (Y-TZP = 98.4% vs. Ti = 97.2%), whilst differed considerably in the maxilla (Ti = 98.4% vs. Y-TZP = 84.4%). Again, all failures occurred during the healing phase owing to increased implant mobility. These findings are consistent with the paper by Depprich et al. [57], where the survival rate of zirconia implants obtained from 17 clinical studies was between 74 and 98% after 12–56 months. Payer et al. [58] followed up for 2 years 19 immediately loaded zirconia implants, reporting a 95% survival rate, as determined clinically and radiographically. These results are in accordance with Oliva et al. [59] who determined the same survival rate at 5 years in 371 patients who received 831 one-piece zirconia implants. Kohal et al. [60] found that immediately restored one-piece zirconia implants have 1-year cumulative survival rate comparable to titanium counterparts. In conclusion, the clinical data currently available for Y-TZP implants may not be sufficient to recommend their routine clinical use. Zirconia, however, may have the potential to be a successful implant material, although this is as yet not fully supported by present

of the tibia of rabbits for 30 months.

68 Dental Implantology and Biomaterial

investigations and further good-quality research is needed.

compared with the monophase ceramics [64] (**Table 2**).

**(AZT)**

**3.1. Background**

**3. Zirconia-toughened alumina (ZTA) and alumina-toughened zirconia**

The demand of structural ceramics has led to an increased interest in Alumina–Zirconia composites for biomedical [21, 61] and dental implant application [62, 63]. Two composite materials can be prepared: ZrO2 reinforced with alumina particles, which is denominated alumina toughened zirconia (ATZ), and Al2O3 reinforced with zirconia particles, which is known as zirconia-toughened alumina (ZTA). Thus, higher fracture values can be reached if These composites benefit from combining the characteristics of Alumina, namely the high hardness and stiffness, with the superior strength and toughness of Zirconia, which improves remarkably the resistance to crack growth [65]. In addition, alumina increases the hydrother‐ mal stability of tetragonal Zirconia phase [65, 66], owing mainly to the formation of a stiff matrix capable to keep the Zirconia particles in a metastable tetragonal state [67], thus acting as mechanical stabilizer. The only commercially used ATZ oral implant was tested both statically and dynamically for its fracture resistance in different simulated oral conditions with satisfying results [68].

The main features of the aforementioned implant are reported in the following table (**Table 3**).


**Table 3.** Physical and mechanical properties of ATZ values refer to Spies et al. (2015) – [68]

Very recently, a complete powder injection molding process was developed to fabricate cylindrical ZTA parts recurring to a binder system made of high-density polyethylene, paraffin wax, and stearic acid. The effects of sintering temperature on shrinkage, relative density, and hardness of the sintered part were taken into account and proved the technology suitable for the production of ZTA parts with sufficient mechanical properties [69]. However, ATZ and ZTA are usually produced through the classic workflows described above for Y-TZP (see Section 2.2).

The favorable mechanical features and the biological safety of different ZTA and ATZ composites have been the object of several studies in the last years [64, 70–78]. Whilst ATZ materials show increased mechanical stability [79] and improved aging resistance versus Y-TZP, still they exhibit a certain degree of aging [79]. ZTA materials display much better aging resistance than both monolithic Y-TZP and ATZ [21, 79, 80]. In a recent work by our research group, both ATZ and ZTA were functionalized with two laminins as a preliminary investiga‐ tion for improving soft tissue healing around implants. The simple adsorption of these two different isoforms was sufficient to induce some of the most important cell kinases in the epithelial cells grown on the surface of the two Alumina–Zirconia composites, supporting the possible advantages of these materials in dental implantology [81]. On this basis, we further studied the behavior of ATZ dental implants treated with a patented hydrothermal process, comparing them to a clinical use titanium surface in a minipig model. Bone healing was assessed through histology and mRNA expression at different time points (8, 14, 28, and 56 days). The most interesting outcome was a statistically significant higher percentage of newly formed bone along ATZ implants, at 56 days, suggesting that the tested material proved to be a promising candidate among the possible ceramic dental implants [82]. Interestingly, by comparing the bone-to-implant contact of moderately roughened ATZ implants (Sa = 1.51 μm) to an anodized titanium standard (Sa = 1.31 μm) in Sprague–Dawley rats, Kohal et al. [83] found that titanium greatly (58%/75%) outperformed the ceramic implant (24%/41%) after a healing period of 14 and 28 days. In addition, at the same time points, the mechanical interlock measured as push-in values increased from 20 to 39 N for titanium and from 10 to 25 N for ATZ. Although the moderately roughened ATZ implants were well accepted in rat bone, their osseointegration process seemed to proceed more slowly than that of anodized titanium.

However, the concerns raised in light of the in vivo data reported by Kohal et al. [51] seemed not to be completely consistent with the promising outcomes of the clinical study conducted by the same research group. Indeed, the cumulative survival rate (94.2%) of one-piece ATZ implants immediately restored with partial fixed prostheses was comparable to that of the loaded titanium implants, in a human clinical trial involving 40 patients after 3 years of observation. In addition to the marginal bone loss (0.79 mm), several soft tissue parameters and patient-reported outcome measures were evaluated suggesting the potential of ATZ for clinical utilization [84]. Notwithstanding the clinical use, little information is still available about the ideal surface treatment that a ceramic dental implant should receive. To better understand whether roughness or hydroxyapatite precipitation capability were more likely to be efficient in terms of surface modifications, we designed a simple in vitro pilot study.

#### **3.2. Material and methods**

#### *3.2.1. Sample preparation*

Two high purity, ready-to-press powders were used to produce the ATZ (ZrO2-20 wt% Al2O3, TZ-3Y20AB, Tosoh, Japan) and ZTA (Al2O3-16 wt% ZrO2, Taimicron, Taimei, Japan) samples. As reported elsewhere [85], specimens were prepared through linear pressuring at 80 MPa followed by cold isostatic pressing at 200 MPa. The process parameters for sintering were as follows: heating up to 700°C at a rate of 50°C/h, followed by a 2-h dwell; heating up to 1500°C at a rate of 100°C/h, followed by a 2-h dwell. The resulting fully dense materials were 12-mm disks with thickness ranging between 4 and 5 mm.

As reported in the diagram below, both ATZ and ZTA discs were either mirror polished with diamond suspension in ethanol with decreasing granulometry to the final surface roughness of <1 micron. Also as-fired samples were used to evaluate the influence of the surface rough‐ ness on the biological response. Subsequently, the samples were either bioactivated with phosphoric acid under hydrothermal conditions (patent numbers: TO2012A000029 and PCT/ IB2013/050425) or left untreated (**Figure 2**).

**Figure 2.** Schematic representation of samples treatments.

#### *3.2.2. Surface characterization*

group, both ATZ and ZTA were functionalized with two laminins as a preliminary investiga‐ tion for improving soft tissue healing around implants. The simple adsorption of these two different isoforms was sufficient to induce some of the most important cell kinases in the epithelial cells grown on the surface of the two Alumina–Zirconia composites, supporting the possible advantages of these materials in dental implantology [81]. On this basis, we further studied the behavior of ATZ dental implants treated with a patented hydrothermal process, comparing them to a clinical use titanium surface in a minipig model. Bone healing was assessed through histology and mRNA expression at different time points (8, 14, 28, and 56 days). The most interesting outcome was a statistically significant higher percentage of newly formed bone along ATZ implants, at 56 days, suggesting that the tested material proved to be a promising candidate among the possible ceramic dental implants [82]. Interestingly, by comparing the bone-to-implant contact of moderately roughened ATZ implants (Sa = 1.51 μm) to an anodized titanium standard (Sa = 1.31 μm) in Sprague–Dawley rats, Kohal et al. [83] found that titanium greatly (58%/75%) outperformed the ceramic implant (24%/41%) after a healing period of 14 and 28 days. In addition, at the same time points, the mechanical interlock measured as push-in values increased from 20 to 39 N for titanium and from 10 to 25 N for ATZ. Although the moderately roughened ATZ implants were well accepted in rat bone, their osseointegration process seemed to proceed more slowly than that of anodized titanium.

However, the concerns raised in light of the in vivo data reported by Kohal et al. [51] seemed not to be completely consistent with the promising outcomes of the clinical study conducted by the same research group. Indeed, the cumulative survival rate (94.2%) of one-piece ATZ implants immediately restored with partial fixed prostheses was comparable to that of the loaded titanium implants, in a human clinical trial involving 40 patients after 3 years of observation. In addition to the marginal bone loss (0.79 mm), several soft tissue parameters and patient-reported outcome measures were evaluated suggesting the potential of ATZ for clinical utilization [84]. Notwithstanding the clinical use, little information is still available about the ideal surface treatment that a ceramic dental implant should receive. To better understand whether roughness or hydroxyapatite precipitation capability were more likely to be efficient in terms of surface modifications, we designed a simple in vitro pilot study.

Two high purity, ready-to-press powders were used to produce the ATZ (ZrO2-20 wt% Al2O3, TZ-3Y20AB, Tosoh, Japan) and ZTA (Al2O3-16 wt% ZrO2, Taimicron, Taimei, Japan) samples. As reported elsewhere [85], specimens were prepared through linear pressuring at 80 MPa followed by cold isostatic pressing at 200 MPa. The process parameters for sintering were as follows: heating up to 700°C at a rate of 50°C/h, followed by a 2-h dwell; heating up to 1500°C at a rate of 100°C/h, followed by a 2-h dwell. The resulting fully dense materials were 12-mm

As reported in the diagram below, both ATZ and ZTA discs were either mirror polished with diamond suspension in ethanol with decreasing granulometry to the final surface roughness

**3.2. Material and methods**

70 Dental Implantology and Biomaterial

disks with thickness ranging between 4 and 5 mm.

*3.2.1. Sample preparation*

Microstructure was studied by means of a scanning electron microscope Zeiss EVO 50 with energy dispersion spectroscopy analyzer for elemental composition detection. Surface roughness was measured with a non-contact profilometer, Talysurf CCI 3000A. The tests were performed in an air-conditioned laboratory, where temperature is kept at 20°C, on a repre‐ sentative surface of 90 μm2 . To quantify the amount of protein adsorbed, fetal bovine serum (FBS) was diluted in phosphate-buffered saline (PBS) at a concentration of 2% and was used to incubate the samples at 37°C for 30 min. After two wash in PBS, the adsorbed protein was eluted from the disks using Tris Triton buffer (10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% Glycerol, and 0.1% SDS) for 10 min. Finally, the total protein amount was quantified using Pierce™ BCA Protein Assay Kit (Life Technologies, Milan, Italy) following the manufacturer's instructions.

#### *3.2.3. Biological response*

Pre-osteoblastic murine cells MC3T3-E1 (ECACC, Salisbury, UK) were used to characterize the biological response in vitro. Cells were maintained in alpha MEM supplemented with 10% FBS (Life Technologies, Milan, Italy), 100 U/ml penicillin, 100 mg/ml streptomycin, under a humidified atmosphere of 5% CO2 in air, at 37°C. To prevent contact inhibition, cells were always passaged at subconfluency. When required, to differentiate MC3T3 cells, the culture medium was supplemented with 10 mM ß-glycerophosphate and 50 ug/ml ascorbic acid.

To examine cell morphology, MC3T3 cells were seeded at a concentration of 5000 cells/well in a 24-well plate. After 1 day, cells were fixed in 4% paraphormaldheyde in PBS. Rodamine– Phalloidin and Dapi (Life Technologies, Milan, Italy) were, respectively, used to stain cytos‐ keleton and cell nuclei, thus evaluating cell adhesion and morphology.

Alkaline phosphatase activity was quantified using the Alkaline Phosphatase Assay Kit (Abcam, Cambridge, UK). Following the manufacturer's instruction, the OD was measured at a wavelength of 405 nm. The calcium deposed within the extracellular matrix was quantified colorimetrically through the Calcium Assay Kit (Cayman Chemical, Michigan, USA). Absorb‐ ance of the lysates was measured at 570 nm.

Data were analyzed recurring to GraphPad Prism6 (GraphPad Software, Inc., La Jolla, CA, USA). Each experiment was repeated at least three times. Statistical analysis was performed using the Student t-test. A p value of <0.05 was considered significant.

### **3.3. Results and discussion**

The success of dental implants is directly related to the bone implant interlock, which can be experimentally evaluated in animal living bone, by histomorphometry and/or biomechanical testing [50]. A moderately rough surface topography is known to positively affect the interfa‐ cial tissue reaction [86]. Surface modification of zirconia and its composites is, however, challenging. Among the roughening techniques used to attain proper bone–implant interfaces, it is convenient to remember the apposition of sintering particles, nano-technology, sandblast‐ ing and acid etching, and laser technology [50, 87–90]. In recent animal studies, in vivo evidence was found that alumina-toughened-zirconia is a suitable candidate for dental implantology [82], which was further supported by very recent clinical data at the University of Freiburg [84, 91]. Following Dohan Ehrenfest's classification [92], surface roughness was moderate (Sa = 1.51 μm) in case of Ziraldent implants or very high (Sa = 5.4 μm) for our research group. Here, the microstructure of ATZ and ZTA was determined by SEM (**Figure 3**).

The materials show an almost defect-free surface, with a homogeneous distribution of both zirconia and alumina. Submicrometric grains are present in both composites, the darkest representing alumina phase. It is noteworthy that similar dimensions can be observed for alumina and zirconia grains only in ATZ material, whilst ZrO2 growth is inhibited by the predominant alumina content in ZTA composite.

**Figure 3.** Scanning electron micrographs of ATZ (A) and ZTA (B).

Surface roughness was measured via profilometry (**Table 4**). As it can clearly be seen, polished and as-fired samples were, respectively, endowed with a very smooth and a highly rough surface, according to the expected values.


**Table 4.** Surface roughness measured via profilometry.

Alkaline phosphatase activity was quantified using the Alkaline Phosphatase Assay Kit (Abcam, Cambridge, UK). Following the manufacturer's instruction, the OD was measured at a wavelength of 405 nm. The calcium deposed within the extracellular matrix was quantified colorimetrically through the Calcium Assay Kit (Cayman Chemical, Michigan, USA). Absorb‐

Data were analyzed recurring to GraphPad Prism6 (GraphPad Software, Inc., La Jolla, CA, USA). Each experiment was repeated at least three times. Statistical analysis was performed

The success of dental implants is directly related to the bone implant interlock, which can be experimentally evaluated in animal living bone, by histomorphometry and/or biomechanical testing [50]. A moderately rough surface topography is known to positively affect the interfa‐ cial tissue reaction [86]. Surface modification of zirconia and its composites is, however, challenging. Among the roughening techniques used to attain proper bone–implant interfaces, it is convenient to remember the apposition of sintering particles, nano-technology, sandblast‐ ing and acid etching, and laser technology [50, 87–90]. In recent animal studies, in vivo evidence was found that alumina-toughened-zirconia is a suitable candidate for dental implantology [82], which was further supported by very recent clinical data at the University of Freiburg [84, 91]. Following Dohan Ehrenfest's classification [92], surface roughness was moderate (Sa = 1.51 μm) in case of Ziraldent implants or very high (Sa = 5.4 μm) for our research group. Here, the

The materials show an almost defect-free surface, with a homogeneous distribution of both zirconia and alumina. Submicrometric grains are present in both composites, the darkest representing alumina phase. It is noteworthy that similar dimensions can be observed for alumina and zirconia grains only in ATZ material, whilst ZrO2 growth is inhibited by the

Surface roughness was measured via profilometry (**Table 4**). As it can clearly be seen, polished and as-fired samples were, respectively, endowed with a very smooth and a highly rough

using the Student t-test. A p value of <0.05 was considered significant.

microstructure of ATZ and ZTA was determined by SEM (**Figure 3**).

predominant alumina content in ZTA composite.

**Figure 3.** Scanning electron micrographs of ATZ (A) and ZTA (B).

surface, according to the expected values.

ance of the lysates was measured at 570 nm.

**3.3. Results and discussion**

72 Dental Implantology and Biomaterial

Interestingly, from **Figure 4**, it can be inferred that the only condition capable to affect significantly the protein adsorption was surface roughness. No statistically significant difference was found among materials (ATZ vs. ZTA) or chemical treatment (hydrothermal cycle present + vs. absent -), although a trend in facilitating protein adsorption could be noted in roughened-treated surfaces.

**Figure 4.** Quantification of adsorbed bovine serum albumin (BSA) by samples, as measured trough BCA assay (see Methods). The rough surfaces significantly increase the amount of adsorbed proteins.

The same trend described for protein adsorption was observed in the other cell-based assays whether they were focusing on the early cell response as in the focal adhesion density (**Figure 5**), or they were dealing with intermediate and late stages of osteogenic differentiation in vitro such as alkaline phosphatase activity (**Figure 6A**) and calcium deposition within the extracellular matrix (**Figure 6B**).

**Figure 5.** Morphology of MC3T3 cells seeded on different surfaces and stained with phalloidin–rhodamine and DAPI to visualize, respectively, the cytoskeleton and nucleus (see Methods). (A) Quantification of focal adhesion density measured by normalizing the number of focal adhesions on cell area (see Methods). (B) The rough surfaces significant‐ ly increase the density of focal adhesion.

**Figure 6.** Colorimetric quantification of ALP activity (A) and calcium deposition (B) (see Methods). The rough surfaces significantly increase the level of either ALP activity (A) and calcium deposition (B).

Although surface chemistry is known to play a role in cueing the biological systems [81], the present experimental data showed that roughened surfaces were more efficient in inducing an osteogenic response in vitro independently of the application of the chemical treatment. In other terms, roughness per se seemed to overpower the effect of the chemical treatment which was deemed bioactive on the ground of the Kokubo tests previously performed (i.e., the capacity to induce hydroxyapatite precipitation) [85]. Within the obvious limits of this experimental setting, our results support the importance of roughening modifications over the chemical treatment.
