Effects of Surface Morphology of ZnAl 2 O 4 Ceramic Materials on Osteoblastic Cells Responses

1 Laboratorio de Bioingenieŕıa de Tejidos, Facultad de Odontologı́a, Universidad Nacional Autónoma de México, Coyoacán, 04510 Ciudad de México, DF, Mexico 2 Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Coyoacán, 04510 Ciudad de México, DF, Mexico 3 Tissue Bioengineering Laboratory, Division of Research and Postgraduate Studies, Faculty of Dentistry, National Autonomous University of Mexico (UNAM), Circuito Exterior s/n, Coyoacan, 04510 Mexico City, DF, Mexico


Introduction
Oxide spinel material is a very large group of structurally related compounds [1], many of which are of considerable technological or geological importance [2].Spinels exhibit a wide range of electronic and magnetic properties.The normal spinel is a typical example of a material with the general formula (X)[Y] 2 O 4 , where X and Y are divalent and trivalent ions, respectively, and the symbols () and [] refer to the 8 tetrahedral coordinated A sites and 16 octahedral coordinated B sites, respectively, within the cubic cell.ZnAl 2 O 4 is an oxide spinel with a close-packed face centered cubic structure and Fd3 m space group symmetry [3].Moreover, its band gap of 3.8 eV makes it transparent for light possessing wavelengths >320 nm; these characteristics allow to use it as a host lattice for applications in thin film electroluminescent displays, mechanooptical stress sensors, and stress imaging devices.On the other hand, this material has good catalytic properties such as cracking, dehydration, and dehydrogenation [4].The spinel zinc aluminates have been widely used as ceramic and as catalytic material in chemical and petrochemical industries [5] and more recently as transparent conductor.Regarding the biological application potentials of this ZnAl 2 O 4 ceramic material in thin films and in bulk are very scarce.The search for bone substitute is still a challenge to researchers.The composition, as well as the topography, of such materials is of importance for determining the biological response to such materials [6].The roughness of materials is considered to be important to predict interfacial behavior at the material-tissue interface and its interaction with the biological environment.Surface roughness influences cell bioactivity, being important in several bone formation stages, including adhesion, proliferation, differentiation, synthesis of bone matrix, maturation, and calcification of the tissue on the materials surface [7][8][9][10].Besides, bioactivity or bioinertness could result in materials with different physical characteristic that could influence biological behavior [11,12].Recently, a number of studies have been carried out to investigate the Zndoped ceramics as biomaterials in bone tissue engineering [13,14].Similar to calcium, zinc has long been recognized as an essential trace element for the proper maintenance of bone growth, with over 85% of the total body zinc residing in bone [15,16].Zn has a stimulatory effect on bone formation, and its deficiency has been associated with retardation and failure of bone growth in animals [17,18].Zn-substituted ceramics were found that modulate the attachment, proliferation, and differentiation of osteoblasts and modulate the activity of bone formation by the cells [19][20][21].In this context, the aim of this study was to investigate the effect of surface morphology of nanostructured thin films of ZnAl 2 O 4 prepared by spray pyrolysis and bulk pellets of polycrystalline ZnAl 2 O 4 prepared by chemical coprecipitation reaction on the biological response of osteoblastic cells in order to evaluate the surface cell adhesion, spreading, cell viability process at in vitro cell culture and compare these results with respect to the different surface morphologies between a nanostructured thin film and a traditional polycrystalline ceramic surface.

Synthesis and Characterization of ZnAl
2 O 4 Material.The ultrasonic spray pyrolysis technique is a well-established process for depositing films.Some advantages of this process are as follow: a high deposition rate, the possibility to coat large areas, its low cost, its ease of operation, and the quality of the coatings obtained.Films of zinc aluminate were deposited by an ultrasonic spray pyrolysis technique described earlier [22].Basically, this technique consists of an ultrasonic generator used to produce a mist from the spraying solution.This mist is carried to a hot substrate placed on a tin bath through a tubing setup using humid air as a carrier gas (10 liters/minute).When the mist of the solution gets in touch with the hot substrate, the solvents in the solution are vaporized producing a solid coating on the substrate.The nozzle in this system is localized approximately 1 cm above the substrate.The spraying solution consisted of 0.05 M zinc acetate and aluminum chloride in deionized water as solvent.The solution flow rate was 3 mL/minute for all cases.The substrate temperature (  ) during deposition was in the range from 300 ∘ C to 550 ∘ C; the substrates used were Corning 7059 glass slides.The deposition time was adjusted (4 to 6 minutes) to deposit films with approximately the same thickness.The thickness of the films studied was about 5 m as measured by a Sloan Dektak IIA profilometer.The chemical composition of the films was measured with a Leica-Cambridge electron microscope Mo.Stereoscan 440, equipped with a Beryllium window X-ray detector, using Energy Dispersive Spectroscopy (EDS).The standard used for the EDS measurements was the Multielement X-ray Reference Standard (Microspec), Serial 0034, part no.8160-53.The surface morphology was analyzed by means of the scanning electron microscopy (SEM) cited above.The crystalline structure features of the deposited films were analyzed by X-ray diffraction (XRD), using a Siemens D-5000 diffractometer with a wavelength radiation of 1.5406 Å (Cuk  ).
A very simple chemical precipitation process was used for the synthesis of zinc aluminate spinel powder.The start materials were Zn (NO 3 ) 2 ⋅ 6H 2 O Sigma-Aldrich (98%) and Al(NO 3 ) 3 ⋅ 9H 2 O Riedel-de Haën at 0.05 M blended in methanol.This simple process consists of three steps: (1) precursor material dissolution in a compatible solvent to form the precursor mixture, (2) solvent evaporation and solute precipitation, and (3) powder annealing.The initial mixture was heat treated at 250 ∘ C for 30 minutes to evaporate the solvent.The chemical agglomerates were grounded in an agate mortar to obtain fine powder, which was compressed to form a small disk with a dimension of 1.2 cm in diameter and a thickness of 0.13 cm.The applied pressure for pellets preparation was 150 Kg/cm 2 .These pellets were annealed at   = 600 ∘ C, during 14 hours in air atmosphere.
The crystalline structure of these pellets was analyzed by X-ray diffraction (XRD) using a Bruker-D8 plus Diffractometer with CuK  radiation at 1.5405 Å.Their chemical composition was measured using Energy Dispersive Spectroscopy (EDS) with a Cambridge-Leica electron microscope mod.Stereoscan 440 was equipped with a Beryllium window Xray detector, and their surface topography was obtained by means of the above-mentioned SEM microscope.

Biological Response
2.2.1.Cell Culture.Biological assays were performed using human osteoblastic cells as reported previously [23].For cell culture, human osteoblastic cells were cultured in 75 cm 2 cell culture flasks containing a Dulbecco's Modified Eagle Media (DMEM), supplemented with 10% fetal bovine serum (FBS) and antibiotic solution (streptomycin 100 g/mL and penicillin 100 U/mL, Sigma Chem.Co).The cell cultures were incubated in a 100% humidified environment at 37 ∘ C in atmosphere of 95% air and 5% CO 2 .Human osteoblastic cells on passage 4-6 were used for all the experimental procedures.In order to perform the in vitro cell response assays, all ZnAl 2 O 4 bulk and 550 ∘ C nanostructured material surfaces were cleaned with distilled water and sterilized by exposure to UV light ( = 254 nm, 300 uW/cm 2 ).

Cell Attachment.
The cell adhesion of human osteoblastic cells onto ZnAl 2 O 4 bulk and thin film nanostructure materials was evaluated using the vibrant cell adhesion assay kit (Molecular Probes).Human osteoblastic cells, cultured in a 75 cm 2 cell culture flask, were washed with phosphatebuffered saline (PBS) and incubated with calcein AM stock solution to a final concentration of 5 × 10 −6 M in serum-free medium for 30 min.After incubation, the cells were washed

Results and Discussion
The surface morphology of ZnAl 2 O 4 coatings deposited on glass substrates is presented in Figure 1.SEM micrographs show the samples deposited at 300 ∘ C Figure 1(a), 450 ∘ C Figure 1(b), and 550 ∘ C Figure 1(c).It is possible to observe rough but continuous coatings with good adherence to the substrate.This figure shows that the surface morphology of the layers depends on substrate temperature.Coatings deposited at 300 ∘ C and 450 ∘ C present some cracks.By increasing the substrate temperature to (500-550 ∘ C), the cracks disappear and a relatively more dense material is reached.These features could be explained because at higher substrate temperature, the deposited radicals are characterized by higher surface kinetic energy, which permits them better accommodation and consequently produces a better processed and compacted material.
At   = 550 ∘ C, the thin film shows a nanogranular morphology with a great quantity of porous regions, which mimics the surface morphology of the human bone.
The chemical composition of the films deposited at substrate temperature of 550 05-0669 [24]).The calculated lattice parameters ( =  =  = 8.0859 Å) for the cubic spinel phase in the films deposited at 550 ∘ C are in agreement with the reported values ( =  =  = 8.0848 Å) [24].Furthermore, it promoted the crystal growth of this material with a preferential (311) orientation normal to the coatings surface.Considerable peaks broadening can be observed due to the nanometric dimension of the grains in the thin film.By using the Debye-Scherrer formula for the broadening fitting curve XRD program, the particle size was evaluated.The average particle diameter was around 20 ± 5 nm, considering that the grains are spheres.
The surface morphology of the polycrystalline ZnAl 2 O 4 pellets sample obtained by chemical coprecipitation process is presented in Figure 3 and its XRD measurements pattern is shown in Figure 4.It is clear that this type of ceramic shows a smooth and homogeneous surface morphology without any observable porous region.Its average grain size is about 8.5 ± 2.0 m.
The biomaterial surface interaction between scaffolds and tissue cells is a significant subject for biomaterials science.Information originating from this interaction is essential to aid the design and fabrication of new biocompatible materials [25].Our results showed that when we culture osteoblastic cells on ZnAl 2 O 4 nanostructured materials, the cell morphology had attached and undergone significant spreading, elongated demonstrating areas, where filopodia had intimately adapted with multiple cellular extensions on the surface of the ceramic (Figure 5(b)).In contrast, osteoblastic cells culture on ZnAl 2 O 4 bulk materials showed neither or small elongation or extension (Figure 5(a)).These morphological results could be supported by the results of cell adhesion values after 4 and 24 h, presented as the cellular percentage of attached cells in relation to control tissue cultures plates.The cellular adhesion as the first step to assess the compatibility of the cell-material interaction surface was 60 to 80% greater on the ZnAl 2 O 4 nanostructured material surface where it should be noted that the adherent values at all-time points were consistently higher when comparing with bulk ceramic surfaces (Figure 6).Statistical analyses indicated that there were statistically significant differences in the cell response, where osteoblastic cells attachment occurred preferred on the rough ceramic surface followed by the smooth surface ceramic.Moreover, it is important to remark that increased cellular attachment obtained on ZnAl 2 O 4 bulk and 550 ∘ C thin film nanostructured materials is a good indicator that the surface is not toxic to the cells.We perform the cell viability test assessed by the MTT assay to confirm it.The results of the MTT assay are presented as the optical absorbance at 570 nm as shown in Figure 7.Both ZnAl 2 O 4 nanostructure and bulk ceramics exhibited excellent biocompatibility.Among the two ceramics, it can be seen that cell viability is always higher on a nanostructured material than a bulk material, where we found high levels of MTT conversion and continue until day 7.This increment is directly proportional to the increase of metabolic active cells on the surface of ZnAl 2 O 4 and inversely proportional to the toxicity effect of the surface topography of the material where significant differences in mean optical density are always presented as confirmed by Student's -test.This increase in adhesion and viability by MTT activity of cells could be favored for the presence of ZnAl 2 O 4 nanoparticle material.These results are in agreement with the idea that topography of extracellular microenvironment can influence cellular responses from attachment and migration to differentiation and production of new tissue [26][27][28][29].Moreover, it has also been reported that surface energy is a more influential surface characteristic on cellular adhesion and proliferation [30,31].So the enhanced cellular adhesion and viability on ZnAl 2 O 4 nanostructure ceramic be due to the positive influence of the component of the surface energy.However, further studies are needed with these materials to fully understand the tissue cell-material interactions.

Conclusion
ZnAl 2 O 4 nanostructure and bulk spinel ceramic have been evaluated for their in vitro biocompatibility to explore their  potential to be used in dental implant and bone substitute applications.The in vitro attachment and morphological and viability responses of osteoblastic cells suggest that nanostructured ceramic appears to be the most conducive to cells compared to the bulk ceramic surface.The results of these studies could lead to a relatively new generation of bioceramics with surface characteristics specific to the needs of individual tissue types as bone or oral cavity.

Figure 4 :
Figure 4: XRD of the bulk ceramic sample annealed at 600 ∘ C.
∘ C was determined by EDS, with the atomic percentages of Zn = 13.6,Al = 27.8,O = 57.1, and Cl = 1.50, which means that we have ZnAl 2 O 4 ceramic compound of stoichiometric composition doped with 1.50 % of chlorine.XRD measurements carried out on the ZnAl 2 O 4 coatings deposited by spray pyrolysis technique are presented in Figures 2(a), 2(b), and 2(c).These XRD patterns are shown for ZnAl 2 O 4 at these three different substrate temperatures: 300 ∘ C, 450 ∘ C, and 550 ∘ C. The zinc aluminate coatings remain in the amorphous state when deposited at substrate temperatures up to 400 ∘ C (Figure 2(a)); as the substrate temperature is increased to 450 ∘ C, some peaks corresponding to hexagonal phase of ZnO (zincite, ICCD Card File no.36-1451) are observed in Figure 2(b).In case of the sample deposited at 550 ∘ C Figure 2(c), only a cubic spinel crystalline phase of ZnAl 2 O 4 (gahnite) was found (ICCD Card File no.
2.2.3.MTT Assay.Cell viability of human osteoblastic cells seeded at concentration of 1 × 10 4 cells/cm 2 onto ZnAl 2 O 4 of the blue formazan product is directly proportional to the number of metabolically active cells.The human osteoblastic cells seeded onto ZnAl 2 O 4 bulk and thin film nanostructure materials at prescribed time were washed with PBS and incubated with fresh cultured medium containing 0.5 mg/mL of MTT for 4 h at 37 ∘ C in the dark.Then, the supernatant was removed and dimethyl sulfoxide (DMSO) was added to each well.After 60 minutes of slow shaking, the absorbance was quantified by spectrophotometry at 570 nm with a plate reader.The culture medium during experimental time was changed every other day with fresh media.2.2.5.Statistical Analysis.Data are presented as mean standard deviation.Statistical analysis was performed on adhesion and MTT assay results using Student's -test, and  value <0.05 was considered significant.