Biocompatible hollow-strut,silica enriched zirconia foams

Abstract

Background:

Porous ceramic biomaterials structures are accepted components in applied research in the field of tissue engineering due to their mechanical properties being closer to structural tissue like bone or other properties related to improved biocompatibility.

Objective:

Hollow-strut, silica enriched zirconia foams were made by replication of polyurethane via impregnation with a suspension of zirconia-particles in polysiloxane.

Methods:

Two-step heat treatment allowed conversion of the precursor structures into hollow-strut ceramic foams which were tested for their biocompatibility using an osteoblast cell line. Further, the material was characterized via different spectroscopic (Raman-spectroscopy, EDX) and imaging (SEM, μCT) methods.

Results:

The material shows open cell porosity with hollow struts and sufficient structural integrity for handling and an expected chemistry as investigated by Raman and EDX spectroscopy. The material further supported cell growth and overall good biocompatibility.

Conclusions:

The investigated composite foam shows promising properties and is potentially interesting as candidate material for future bone tissue engineering applications.

Keywords

Zirconia silica foams biocompatibility spectroscopy

1. Introduction

Porous ceramics are widely accepted in applied biomaterials research and tissue engineering [1] and when compared to other biomaterials like polymers and metals, have significantly higher compressive strength and hardness.

Here, a multitude of ceramic materials have been established as porous foams and include calcium phosphates [2], alumina [3], zirconia [4], titanium dioxide [5], bio-glasses [6] and glass ceramics [7,8]. While calcium phosphates and some bio-glass compositions are potentially biodegradable at least to some extent, the other systems are deemed non-degradable due to the strong chemical bonds. However, mechanical wear and prolonged corrosion can result in failure even of ceramics. Here, tetragonal stabilized zirconia shows an exceptional behaviour, as it features both high mechanical toughness due to its ability known as transformation toughening [9–12], but which is similarly effected by prolonged watery corrosion altering significantly the stability by transforming it into the monoclinic polymorph [13]. Nevertheless, the overall advantageous set of properties including its biocompatibility make zirconia based ceramics ideal candidates in biomaterials science.

In this regard, the combination of a zirconia matrix with silica or glass has been investigated for example by Della Bona et al. [14].

In the present research, silica enriched zirconia foams are made by replication of polyurethane (PU) structures with a suspension of zirconia in polysiloxane. Subsequently, this porous polymeric composite was initially heat treated in argon atmosphere to allow for decomposition of the polyurethane/polysiloxane matrix into carbon species. Following, the porous construct was sintered in ambient atmosphere to achieve the silica enriched zirconia structure.

The resultant porous composite scaffold was optimized in regard of its porosity and was tested for its performance in vitro. The ceramic scaffold was further characterized via different spectroscopic (Raman-spectroscopy, EDX) and imaging (SEM, μCT) methods.

2. Materials and methods

2.1. Synthesis of zirconia-silica composite foams

The composite foam samples were prepared from two different PU foams with two different pore sizes (characterized by the number of pores per inch (ppi) as 15 ppi and 10 ppi) which were impregnated with a suspension of zirconia (3 mol% Y₂O₃) in a RTV-2-polysiloxane (Köraform, Alpina Technische Produkte GmbH, Germany). The principal processing methodology is depicted in Fig. 1.

Fig. 1.

Processing schematic for the synthesis of zirconia-silica composite foams. i) Casting of ZrO₂-silicone slurry onto a PU-foam and impregnation by kneading until full polymerization. ii) Two-step thermal treatment of the impregnated foam in argon atmosphere (up to 500°C) and in ambient atmosphere (up to 1550°C).

In detail, 10 g zirconia powder was mixed with 10 g of component A of the RTV-2-polysiloxane. Afterwards this suspension was mixed with component B of the RTV-2-polysiloxane together with a retardant (1 wt%, Alpina Technische Produkte GmbH, Germany), to slow the crosslinking speed. This mixture was used to impregnate the two different PU foams (dimension: $15 mm \times 15 mm \times 15 mm$ each) by thorough kneading until the silicone starts to consolidate via crosslinking. During this process, it was carefully observed that all pore-struts were completely covered and that the open porosity of the PU foams was not closed by material from the zirconia-RTV-2-polysiloxane suspension. To increase the surface area of the still sticky material, the foams were thoroughly agitated in a Falcon tube with zirconia (3 mol% Y₂O₃) powder.

Samples were heat-treated in a prototype dental oven (Denta-Star M2plus, Thermos-Star, Germany) as follows. Heating in argon-atmosphere in a double chamber SiC-system filled with 1 mm diameter zirconia spheres as oxygen getter and as support for the composite foams (heating rate 100 k/h until 500°C, holding time at 500°C: 6 h followed by heating to 1000°C with 180 k/h). After cooling to room temperature, the samples were removed from the SiC-chamber and placed into the same oven for sintering in oxygen-atmosphere in an alumina crucible on 1 mm diameter zirconia spheres as supports (heating rate 180 k/h until 1550°C, holding time at 1550°C: 6 h) followed by controlled cooling to 650°C with 600 k/h and finally cooling to room temperature.

One sample which was used for in vitro cell cultivation was processed using a different set of ovens due to better aseptic handling. For initial heating in argon-atmosphere (heating rate 100 k/h until 500°C, holding time at 500°C: 6 h) a dental oven was used (Centurion, Dentsply, Germany). Further heating in an alumina crucible with zirconia spheres as support was conducted using a high temperature oven (model HT 04/17, Nabertherm, Germany) at a heating rate of 180 k/h until 1550°C (holding time: 6 h) followed by controlled cooling to room temperature with 600 k/h. The alumina crucible with the sample was transferred aseptically to a clean bench (Thermo Fisher Scientific, Germany) for further sterile handling and cell seeding.

2.2. Cell culture

According to previously published protocols [15], a human osteoblast (CAL-72) cell line was used for the in vitro biocompatibility testing of one zirconia-silica composite scaffold (15 ppi). The CAL-72 osteoblast cell line was originally derived from an osteosarcoma [16]. Briefly, cells were grown to confluence in DMEM culture medium (Biochrom AG, Germany) supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin (P/SA 2212, 10,000 U/ml, Biochrom AG, Germany). Cells were harvested by trypsinization using trypsin/EDTA (0.05%/0.02%) and were resuspended in the cell culture medium, resulting in approximately $10^{6} cells/ml$ . The cell suspension was pipetted onto the composite foam previously positioned in one well of a 6-well plate already containing 2 ml medium (DMEM, supplemented as above). The cell seeded composite foam was cultivated for one week in an incubator (Binder, Germany) at 37°C and 5 CO₂ with medium exchange every other day.

2.3. Light microscopy, scanning electron microscopy and EDX

Light microscopic investigations were performed using a Keyence VHX 100 digital light microscope (Keyence Deutschland GmbH, Germany) due to the high depth of focus as compare to other light microscope systems.

Scanning electron microscopy (SEM) and electron dispersive X-ray analysis (EDX) were conducted according to previously published protocols [17–19] using a CamScan Series 2 SEM, coupled with an EDX detector (EDX Type ECON4/CamScan149, EDAX Inc., USA) at 20 keV. Briefly, samples were glued to aluminum-sample holders (Ø: 12 mm) with conductive carbon-paste and were sputter coated with carbon in a Balzers SCD050 sputter coater (BALTEC, USA) in argon atmosphere at $1.0 \times 10^{- 2} mbar$ . Samples were sputter coated multiple times from different angles to achieve good conductivity of the porous foams.

SEM images were saved as TIFF-files, while EDX data was saved as ASCII-data and analyzed using the Software OriginPro (OriginLabs, USA).

2.4. Fluorescence microscopy

Cell vitality and growth were characterized by fluorescence microscopy using a Leica DM 4000M microscope (Leica Microsystems, Switzerland) with Leica PL Fluotar objectives (2.5× and 5×). For vital fluorescence staining, a combination of fluorescein diacetate (FDA) and ethidium bromide (EB) was used with Leica N2.1 fluorescence cubes according to previously published protocols [20]. A Leica DFC 320 digital camera in combination with Leica IM1000 image acquisition software was used for image acquisition as JPEG-files with software calibrated micrometer bars.

2.5. Raman spectroscopy

Raman microscopy was conducted according to previously published protocols [15] using a WITec alpha300AR Raman microscope system (WITec GmbH, Germany) to distinguish between the different components. Excitation was performed at room temperature at 532 nm. The characteristic Raman shifts for tetragonal, monoclinic and cubic zirconia were compared to the values as discussed in Zehbe et al. and Phillippi et al. [11,21].

2.6. Tomography

Three dimensional tomography data was recorded using a lab-based polychromatic X-ray source from a micro-computed-tomography system (Skyscan1172, Bruker, Kontich, Belgium). To obtain a high contrast in the recorded data, a source voltage of 100 kV and a source current of 100 μA were used in conjunction with an Al/Cu-filter.

Data acquisition was performed using a Hamamatsu C9300 11Mp camera with a pixel size of 8.7 μm and which was exposed for 350 ms. Sample rotation was conducted in steps of 0.2° for 360°. Data was recorded as TIFF-files in 16 bit colour depth.

Projection data was normalized using ImageJ [22] and was reconstructed using the software NRecon (BrukerCT, Kontich, Belgium). The reconstructed volume data was processed using the software ImageJ and Voreen (University of Münster, Germany). The relevant data was saved as cross-sectional views and rendered three-dimensional views displaying the highly absorbing granular ZrO₂-particles in white coloration and the lower absorbing silica matrix in blue. The effective voxel size of the reconstructed data was 17.97 μm.

Fig. 2.

Light microscopic representations of different zirconia-silica composite foams: (a) 15 ppi foam, (b) 10 ppi foam and (c) polished cross-section of a 10 ppi foam.

Fig. 3.

SEM representations of a 10 ppi foam with powder (a) and in higher magnification (b). SEM representation of a single foam cell from a 15 ppi foam (c) and higher magnifications showing the hollow strut structure (d), (e).

3. Results

3.1. Structure and composition

Light microscopic characterization of the silica enriched zirconia composite foams shows overall similar results for the two different pore sizes (Fig. 2(a), 15 ppi and Fig. 2(b), 10 ppi). As can be seen from these overview images, but also in cross sectional view (Fig. 2(c), exemplarily for the 15 ppi foam), the foam structures feature a granular surface consisting of sintered, connected and round shaped zirconia particles. Further, Fig. 2(c) shows the supposedly silica rich component connecting the void space between the zirconia particles with hollow struts resulting from the burn-out of the original PU-foam. Finally, the structure of the PU foam is well preserved and only a minimal amount of pores has been closed due to the kneading process. Matching results from SEM imaging (Fig. 3(a)–(e)) substantiate the particulate surface structure for both the 10 ppi (Fig. 3(a) and (b)) and the 15 ppi foam (Fig. 3(c)/single foam cell). Higher magnifications clearly show the previously observed hollow strut nature of the material (Fig. 3(d) and (e)).

Figure 4 displays the chemical characterization via EDX spectroscopy (Fig. 4(a)) and Raman spectroscopy (Fig. 4(b)). While EDX-spectroscopy clearly shows the presence of the composing elements Si, Zr and Y (with traces of Hf), the lighter elements (O, C) are not distinguishable. The compositional elements are similarly present after the first thermal treatment in argon atmosphere and after the second heat treatment in ambient (oxygen rich) atmosphere.

Fig. 4.

(a) EDX analyses showing the dominant compositional elements Si, Zr and Y (with some Hf impurities). (b) Raman spectrum showing the tetragonal stabilized ZrO₂ (filled peaks) and undefined SiO_x (peaks at: 1240 cm⁻¹, 1340 cm⁻¹, 1430 cm⁻¹, 1540 cm⁻¹).

In this regard, Raman spectroscopy clearly shows the presence of tetragonal zirconia according to the characteristic Raman shifts as reported in [11,21]. Raman shifts at 1240 cm⁻¹, 1340 cm⁻¹, 1430 cm⁻¹ and 1540 cm⁻¹ cannot be linked to specific compounds but supposedly indicate non-stoichiometric silica species.

3.2. Volumetric imaging

The volumetric representation of one 15 ppi composite foam is displayed in Fig. 5(a)–(c) as rendered volume data, showing the strong X-ray absorbing fused zirconia particles coloured white and the lesser absorbing silica species coloured pale blue.

Fig. 5.

Rendered tomographic representations of a 15 ppi composite foam (a) structural overview, (b) higher magnification showing the granular ZrO₂-particles (white) in the silica matrix (blue), (c) high magnified field of view showing a single foam cell and (d) cross section of the composite foam with a high magnified field of view showing the hollow strut structure (i).

Figure 5(b) and (c) shows higher magnified volumetric representations, with Fig. 5(c) displaying a single foam cell. The data shows a mostly open pore-structure corresponding to findings from SEM imaging.

Figure 5(d) shows exemplarily the sliced grey value data with brighter domains corresponding to the white coloured zirconia particles and the more greyish silica species (coloured pale blue in the rendered volumetric data). The magnification in (e) shows the hollow strut nature of the porous scaffold.

3.3. In vitro characterization

One single silica enriched zirconia composite foam was investigated after initial seeding with $10^{6}$ cells (osteoblast-like, type CAL-72) and further cultivation for one week using vital fluorescence staining (FDA & EB).

Cells were spreading evenly across the foam struts, covering all structures of the material and showing predominantly vital cells (FDA positive: green fluorescence) and no visible dead cells (EB positive: red fluorescence) indicating good biocompatibility of the material and support of cell growth over the whole cultivation period of one week (Fig. 6(a) and (b)).

Fig. 6.

(a) Vital fluorescence (FDA & EB) stain of CAL-72 osteoblast-like cells adhering on the composite foam surface (green: vital cells, red: non-vital cells). (b) Magnified region of interest.

4. Discussion

In this study, we have prepared and investigated a silica enriched, hollow strut zirconia foam for possible application in tissue engineering. The porous material was synthesized by replication of PU foams using a suspension of tetragonal stabilized zirconia particles in a RTV-2-polysiloxane followed by a two-step heat treatment.

The resulting material shows a high open porosity resembling closely the original PU foam but featuring a hollow strut structure from the burn-out of the replicated polymer. Further, the foam surface appears highly granular in both light microscopy and electron microscopy and is apparently made up of fused zirconia particles. Chemical characterization by EDX and Raman spectroscopy indicates the expected chemistry based on tetragonal zirconia particles and a silica enriched environment.

Finally, biocompatibility has been demonstrated using a human-derived osteoblast-like cell line (CAL-72) which demonstrates cell spreading over the foam surface showing good excellent vitality as indicated by a fluorescent live-dead assay (FDA & EB staining). A relevant clinical application of this kind of material is most probable in the fields of dental or orthopaedic implants. A good review on future perspectives of porous biomaterials for dental and orthopaedic applications is presented by Mour et al. [23]. Next research steps are envisioned to be preclinical testing, adapting protocols which were previously established by some of us in bone tissue engineering [24].

5. Conclusion

In conclusion, the composite scaffold appears to be an ideal substrate for the in vitro cultivation of osteoblast-like cells and therefore can be a candidate material for future bone tissue engineering applications and dental or orthopaedic implants which rely on a porous bone-like structure and a biocompatible surface chemistry.

Footnotes

Acknowledgements

The authors would like to thank Dr. Paul Zaslansky and Margret Dilger-Rein from the Charité Berlin for the acquisition of the X-ray μCT data and technical assistance in cell cultivation. The authors further acknowledge the support of the Technische Universität Berlin and the University of Potsdam.

Conflict of interest

The authors have no conflict of interest to report.

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