**3. Biocomposites**

236 Sintering of Ceramics – New Emerging Techniques

Microhardness measurements were carried out using the micro tester FM7 with 100g load.

Wettability research test of glass system to plate substrates from submicrocrystalline sintered corundum carried out at high temperature microscope MH02 by sessile – drop method showed that the glass FB3 was not gasified, there was no significant change in the

a) 110°C a) 904°C a) 1010°C Fig. 10. Wettability of glass FB3 system on the substrate from submicrocrystalline sintered

For further research the submicrocrystalline sintered corundum milled for 20 hours and the glass FB3 system were chosen because of the obtained value of specific surface area of

The results show that these are hard glass systems within 5.5 – 6.0 GPa hardness.

dimensions of the swelling and contact angle theta was about 450.

Fig. 9. DTG of glass FB2 system with gas analysis.

corundum, a) 110°C, b) 904°C, c) 1010°C.

corundum and the properties stability of glass.

The composites containing a matrix from submicrocrystalline sintered corundum and bioglass of Ca0 – Si02 – P205 – Na20 system (FB3) in 10, 20 and 30 wt.%, obtained by powder metallurgy technique, in the process of free sintering in air atmosphere, in the electric furnace. Bioglass of the Ca0 – Si02 – P205 – Na20 system was obtained by fritting process at 1350 oC using an electric furnace.

The composites were obtained with two techniques:


Samples of small (Ø10x2) and large (Ø16x5) size were pressed on the screw press, and after they were isostatically densified or not. The heat treatment was performed without the mould in an electric furnace in air, according to the established characteristics of isothermal soak, at maximum temperature at the time of 2 hours.

Phase composition of biocomposite samples was identified using X – ray diffractometer August Siemens Type D 500 Cristal Reflex with copper lamp with monochromatic radiation.

The phases composition were determined for the samples doped with 10 wt.% bioglass admixture isostatic densification or without densification (w1ssc), the identity of the spectra was found in both cases, suggesting the identity of the phase composition of the samples, regardless of the method of obtaining. In samples following phases were identified : alpha and kappa – Al203 (kappa in the trace amounts), anortit – aluminum calcium silicate – CaAl2 (Si04)2, sodium aluminum silicate NaAlSi04 and quartz Si02.

Fig. 11. The XRD spectra for w1ssc samples a) with isostatic densification, b) without densification.

Biocompatible Ceramic – Glass Composite –

Manufacturing and Selected Physical – Mechanical Properties 239

SiO2 hexagonal a = 4,913

CaO Face – centered cubic a = 4,797 NaAlSiO4 cubic a = 7,37

Al2O3 rhombohedral a = 4,758

Microscopic observations with the electron microscopes Jeol JSM 6460 LV type were performed in high vacuum (~ 1,3 x10-3Pa) at 20 kV accelerating voltage, magnification 20x, 100x and 1000x using of BEC image. Microscopic observations carried out with the scanning electron microscope revealed the differences in the microstructure of samples after isostatic densification or without densification. Microstructures of composites contained grains of irregular shape and varying dimensions and pores. The dimensions of grains were in the range below 1 µm to several µm. Leeks (pores) had a varied shape and dimensions of the order of several micrometers. Samples obtained without densification were more porous; but having more smaller pores (mesopores). Isostatically densified samples had dense microstructure with a small amount of larger pores. Photos of samples formed by various

a) b)

Fig. 14. The SEM observations of the sample W1ssc, magn. 1000x; a) with isostatic

MgAl11LaO19 hexagonal a = 5,582

P2O5 Face centered orthorhombic

CaAl2(SiO4)2 triclinic

Table 4. Crystalographic identification of phase composition.

techniques are presented in Fig. 14, 15 under magnification 1000x.

densification, b) without densification.

Phase Strukture Crystalographic parameters

c = 5,405

c = 21,942

a = 16,3 b = 8,14 c = 5,26 a/b = 2,00246 c/b = 0,64619

c = 12,991

a = 8,21 b = 12,95 c = 14,16 a/b = 0,63398 c/b = 1,09344

Fig. 12. Identification of XRD spectra for w1ssc isostatically densified sample.

Additionally were made the samples with 20, 30 wt.% glass admixture without isostatic densification. It was found, that the phase composition did not change very much.

Fig. 13. The XRD spectra for w1ssc, w2ssc, w3ssc samples without densification.

Fig. 12. Identification of XRD spectra for w1ssc isostatically densified sample.

densification. It was found, that the phase composition did not change very much.

Fig. 13. The XRD spectra for w1ssc, w2ssc, w3ssc samples without densification.

Additionally were made the samples with 20, 30 wt.% glass admixture without isostatic


Table 4. Crystalographic identification of phase composition.

Microscopic observations with the electron microscopes Jeol JSM 6460 LV type were performed in high vacuum (~ 1,3 x10-3Pa) at 20 kV accelerating voltage, magnification 20x, 100x and 1000x using of BEC image. Microscopic observations carried out with the scanning electron microscope revealed the differences in the microstructure of samples after isostatic densification or without densification. Microstructures of composites contained grains of irregular shape and varying dimensions and pores. The dimensions of grains were in the range below 1 µm to several µm. Leeks (pores) had a varied shape and dimensions of the order of several micrometers. Samples obtained without densification were more porous; but having more smaller pores (mesopores). Isostatically densified samples had dense microstructure with a small amount of larger pores. Photos of samples formed by various techniques are presented in Fig. 14, 15 under magnification 1000x.

Fig. 14. The SEM observations of the sample W1ssc, magn. 1000x; a) with isostatic densification, b) without densification.

Biocompatible Ceramic – Glass Composite –

corundum (3,887g/cm3) and glass FB3 system (2,564 g/cm3).

Manufacturing and Selected Physical – Mechanical Properties 241

Studies of real density of the sample dreal (Table 5) showed that, regardless of the method of preparation, with increasing glass content in the composite the real density decreases (from 3,615 to 3,474 g/cm3). It resulted from the different density of submicrocrystalline sintered

The results of determining the apparent density and the skeleton of both open and closed pores became evident that the apparent density decreases with increasing glass content, in both cases of isostatic densification or without densification, but the value of the apparent density of isostatically densified samples was higher than of samples without densification, what resulted from the process of obtaining . Based on density measurements of helium and the actual porosity of the closed set Pz results showed that in the test samples there were present opened pores and closed pores were in negligible volume. Specific surface samples with increasing SBET bioglass content decreased in both cases (samples isostatically densified or without densification). The values of SBET in the 0,5 to approximately 2,0 m2/g were testified by low content of mesopores which volume ranged from 0,001 to 0,005 cm3/g.

Visible is that the total porosity increased with increasing bioglass content for both isostatically densified samples (from 0,146 to 0,197cm3/g) and without densification (from 0,161 to 0,214 cm3/g) containing mainly macropores with dimensions greater than 0.1 microns, which

Measurements of surface area (SBET) were performed using the multifunctional apparatus (ASAP 2010, Micromeritics American company) for measuring surface area and porosity. The specific surface area was determined by physical SBET nitrogen adsorption at liquid nitrogen temperature (77K) from the equation Brunauer – Emmet – Teller (the theory of multilayer adsorption). Before the measurement surfaces of the test samples were subjected to desorption at temperature 1050oC, in vacuum and flushing with pure helium. Sample degassing time was

The specific surface area calculations based on data from the adsorption isotherms of the relative pressure range p/p0 from about 0,06 to about 0,20% and the volume and

Geometric structures of the biocomposites samples surface were determined using the TOPO 01vP profilometer, by measuring the surface topography parameters (Ra, Rz, Rt), the image of 2D and 3D ,profile material rate and amplitude distributions of the ordinates.

The geometric structure of the surface isostatically densified or without densification samples (w1ssc) have been evaluated by measuring surface topography performed using TOPO 01vP profilometer developed and produced in IZTW. There were defined the basic parameters of roughness (Ra, Rz, Rt). In the isostatically densified sample porosity effect on profile was visible (few large cavities- inequality) .This was confirmed by image analysis of 2D and 3D. Participation of the linear bearing and amplitude distribution of the ordinate

The profile roughness of sample without densification has more blurred shape, with more small pits and a few large inequalities compared with the profile of the isostatic densification sample. It was testified by the image analysis of 2D and 3D. The amplitude

distribution of the ordinate showed the presence of inequality – type cavities.

resulted from an increased quantities of bioglass mainly having opened pores.

about 8 hours. Surface degassing state was controlled in automatic mode.

dimensions of the mesopores were calculated using p/p0 of 0,97%.

was shifted toward negative values.

Fig. 15. The SEM observations of the sample W2ssc, magn. 1000x; a) with isostatic densification, b) without densification.

Measurements of the real density (dreal) – of powder samples and helium density (on tablets Ø 16x5mm) were performed using helium pycnometer AccuPyc1330 Micrometrics company. Before the relevant measurements of the samples were initially desorbed by a 10 – fold pure helium flushing. Five parallel measurements were done for each sample. The results were used to calculate the closed porosity. Measurements of apparent density (dap) and total porosity (Pc) were carried out using GeoPyc density analyzer, model 1360 manufactured by Micrometrics. Ten simultaneous measurements were made for each sample. Apparent density (g/cm3), the volume of pores in the material Vc (cm3/g) and total porosity (%) were determined.


Table 5. Density and porosity of biocomposite samples.

a) b)

Measurements of the real density (dreal) – of powder samples and helium density (on tablets Ø 16x5mm) were performed using helium pycnometer AccuPyc1330 Micrometrics company. Before the relevant measurements of the samples were initially desorbed by a 10 – fold pure helium flushing. Five parallel measurements were done for each sample. The results were used to calculate the closed porosity. Measurements of apparent density (dap) and total porosity (Pc) were carried out using GeoPyc density analyzer, model 1360 manufactured by Micrometrics. Ten simultaneous measurements were made for each sample. Apparent density (g/cm3), the volume of pores in the material Vc (cm3/g) and total

> W2 without dens.

3,5296 ±0,0193

3,5010 ±0,0167

2,1564 ±0,0070

Vpores, cm3/g 0,166 0,149 0,178 0,160 0,215 0,198 Vmacro, cm3/g 0,161 0,146 0,176 0,159 0,214 0,197

Vmezo,cm3/g 0,005 0,003 0,002 0,001 0,001 0,001

SBET, m2/g 1,99 1,45 1,02 0,88 0,89 0,50

P, % 36,9 35,9 38,9 36,2 43,5 41,2

W2 isostat. dens

3,5296 ±0,0193

3,5237 ±0,0030

2,2516 ±0,0097

W3 without dens.

3,4743 ±0,0252

3,3942 ±0,0249

1,9637 ±0,0044

W3 isostat. dens

3,4743 ±0,0252

3,4319 ±0,0402

2,0442 ±0,0052

Fig. 15. The SEM observations of the sample W2ssc, magn. 1000x; a) with isostatic

W1 isostat. dens.

3,6150 ±0,0050

3,5457 ±0,0109

2,3179 ±0,0052

densification, b) without densification.

porosity (%) were determined.

dreal, g/cm3 3,6150

dhel, g/cm3 3,6684

dapperend, g/cm3 2,2821

W1 without dens.

±0,0050

±0,0089

±0,0054

Table 5. Density and porosity of biocomposite samples.

Sample/ parameter Studies of real density of the sample dreal (Table 5) showed that, regardless of the method of preparation, with increasing glass content in the composite the real density decreases (from 3,615 to 3,474 g/cm3). It resulted from the different density of submicrocrystalline sintered corundum (3,887g/cm3) and glass FB3 system (2,564 g/cm3).

The results of determining the apparent density and the skeleton of both open and closed pores became evident that the apparent density decreases with increasing glass content, in both cases of isostatic densification or without densification, but the value of the apparent density of isostatically densified samples was higher than of samples without densification, what resulted from the process of obtaining . Based on density measurements of helium and the actual porosity of the closed set Pz results showed that in the test samples there were present opened pores and closed pores were in negligible volume. Specific surface samples with increasing SBET bioglass content decreased in both cases (samples isostatically densified or without densification). The values of SBET in the 0,5 to approximately 2,0 m2/g were testified by low content of mesopores which volume ranged from 0,001 to 0,005 cm3/g.

Visible is that the total porosity increased with increasing bioglass content for both isostatically densified samples (from 0,146 to 0,197cm3/g) and without densification (from 0,161 to 0,214 cm3/g) containing mainly macropores with dimensions greater than 0.1 microns, which resulted from an increased quantities of bioglass mainly having opened pores.

Measurements of surface area (SBET) were performed using the multifunctional apparatus (ASAP 2010, Micromeritics American company) for measuring surface area and porosity. The specific surface area was determined by physical SBET nitrogen adsorption at liquid nitrogen temperature (77K) from the equation Brunauer – Emmet – Teller (the theory of multilayer adsorption). Before the measurement surfaces of the test samples were subjected to desorption at temperature 1050oC, in vacuum and flushing with pure helium. Sample degassing time was about 8 hours. Surface degassing state was controlled in automatic mode.

The specific surface area calculations based on data from the adsorption isotherms of the relative pressure range p/p0 from about 0,06 to about 0,20% and the volume and dimensions of the mesopores were calculated using p/p0 of 0,97%.

Geometric structures of the biocomposites samples surface were determined using the TOPO 01vP profilometer, by measuring the surface topography parameters (Ra, Rz, Rt), the image of 2D and 3D ,profile material rate and amplitude distributions of the ordinates.

The geometric structure of the surface isostatically densified or without densification samples (w1ssc) have been evaluated by measuring surface topography performed using TOPO 01vP profilometer developed and produced in IZTW. There were defined the basic parameters of roughness (Ra, Rz, Rt). In the isostatically densified sample porosity effect on profile was visible (few large cavities- inequality) .This was confirmed by image analysis of 2D and 3D. Participation of the linear bearing and amplitude distribution of the ordinate was shifted toward negative values.

The profile roughness of sample without densification has more blurred shape, with more small pits and a few large inequalities compared with the profile of the isostatic densification sample. It was testified by the image analysis of 2D and 3D. The amplitude distribution of the ordinate showed the presence of inequality – type cavities.

Biocompatible Ceramic – Glass Composite –

MC3T3 – E1 Subclone 14 CRL 2594 line.

*Cell lines.* Two types of cell lines were studied:

**4.1 Materials and methodology** 

Manufacturing and Selected Physical – Mechanical Properties 243

The usefulness verification of these research substrates (w1ssc, w2ssc, w3ssc) for cell culture were checked for short – term culture of human skin CCL line and mouse preosteoblasts

*Bioglass sterilization.* The surfaces used for cell growing were w1ssc, w2ssc, and w3ssc. Before seeding the cells, the following protocol for sterilization has been applied. The samples of the bioglass composite (10 mm in diameter and 2 mm in thickness) were immersed in the 70 % alcohol solution for 12 hours. Afterwards, each side of the sample was exposed for 2 hours to UVC light (wavelength of 245 nm), which was provided by a germicidal lamp from a laminar flow chamber (Nuaire Nu 425), at average intensity of 0.1 mW/cm2 at the working

• human skin fibroblasts (CCL-110, LG Promochem) were cultured in DMEM (Dulbecco's Modified Eagle Medium, Sigma) containing 5 % of fetal bovine serum and 1 % mixture solution of antibiotics (streptomycin, neomycin and penicillin). They were grown at 37ºC in an incubator (NUAIRE, USA) providing 95% air / 5% CO2 atmosphere. Initially, cells were grown in a culture flask (Saarstedt) and when they formed semiconfluent monolayer, they were trypsinized using 0.25% trypsin/EDTA solution

• mouse preosteoblasts MC3T3 – E1 Subclone 14 (CRL-2594, LG Promochem). Cells were cultured in Alpha Minimum Essential Medium with ribonucleisides, supplemented with 10% fetal bovine serum (LG Promochem). They were grown at 37ºC in an incubator (NUAIRE, USA) providing 95% air/ 5% CO2 atmosphere. Analogously to fibroblasts, cells were grown in a culture flask (Saarstedt) and when they formed semiconfluent monolayer, they were trypsinized using 0.25% trypsin/EDTA solution

*Phalloidin staining.* To visualize the organization of actin filaments, cells were stained and imaged using fluorescent microscope (the same protocol was applied independently of the cell type). First, cells grown on bioglass surfaces, were fixed with 3.7% paraformaldehyde dissolved in the PBS buffer for 10 minutes, followed by rinsing them twice in the PBS buffer (Phosphate Bufered Saline, Sigma), permeabilization with 0.1% Triton X-100 solution in the PBS buffer. Then, they were again rinsed in the PBS buffer (3x3 minutes). The actin filaments were stained using the solution containing phalloidin labeled with Alexa Fluor 488 (1:200, in PBS buffer, Molecular Probes) for 30 minutes incubation at room temperature. Next the excess of dye was removed by rinsing bioglass surfaces in the PBS buffer. The wet surfaces

*Fluorescence microscopy.* Fluorescence is the ability of organic or inorganic specimen to absorb and subsequent emit of light. The basic function of a fluorescence microscopy is to irradiate the sample with a desired and specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light. In a properly configured microscope, only the emission light should reach the eye or detector so that the resulting fluorescent

with stained cells were placed on the microscope slide and immediately imaged.

plane. Such sterilized bioglass samples were used immediately for cell growth.

(Sigma) and placed into bioglass surfaces for 96 h and 360 h.

(Sigma) and placed into bioglass surfaces for 96 h and 360 h.

**4. Short term culture of the fibroblast human skin CCL 110 line and mouse** 

**preosteoblasts MC3T3 – E1 Subclone 14 on these substrates** 

Fig. 16. The image of 2D and 3D, along with a profile material rate and amplitude distribution of the ordinates for isostic densification w1ssc sample.

Fig. 17. The image of 2D and 3D, along with a profile material rate and amplitude distribution of the ordinates without densification w1ssc sample.
