**4.1 Materials and methodology**

242 Sintering of Ceramics – New Emerging Techniques

Fig. 16. The image of 2D and 3D, along with a profile material rate and amplitude

Fig. 17. The image of 2D and 3D, along with a profile material rate and amplitude

distribution of the ordinates without densification w1ssc sample.

distribution of the ordinates for isostic densification w1ssc sample.

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

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


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

*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

Biocompatible Ceramic – Glass Composite –

surfaces W1ssc, W2ssc, and W3ssc.

Isostatic

W1ssc Yes fibroblasts

W1ssc No fibroblasts

W2ssc Yes fibroblasts

W2ssc No fibroblasts

W3ssc Yes fibroblasts

W3ssc No fibroblasts

Composite's surface

respectively).

W1ssc and W3ssc was comparable.

surfaces are placed.

Manufacturing and Selected Physical – Mechanical Properties 245

The round cells denotes the cells that starts to detach from the investigated surface. In general, cells spread and grow on rigid substrates if the provided conditions enable them to preserve normal functioning (in such conditions fibroblasts turn into spindle-shaped cells) but they retract, and become round on non-biocompatible ones. The table 6 presents the summary of shapes observed for both fibroblasts and osteoblasts cultured on the studed

densification Cell type Cell shape

spindle-like and round spindle-like

> round spindle-like

spindle-like and round spindle-like

> round round

round spindle-like

spindle-like spindle-like and round

osteoblasts

osteoblasts

osteoblasts

osteoblasts

osteoblasts

osteoblasts

Table 6. Summary of cellular shapes cultured on the different composite's surfaces.

To estimate the number of cells present on composite surface, the percentage of cells present on a given sample type was calculated as a ratio of cells present on the studied surface divided by the number of cells cultured on a glass coveslip (2500 and 4800 cells on a glass rounded coveslip with diameter of 10 mm for human skin fibroblats and mouse osteoblasts,

Figure 19 a and b shows the comparison between the percentage of cells attached to composite surfaces (W1ssc, W2ssc, W3ssc) after 96-hour of growth in culture conditions. In case of fibroblasts, the composite surfaces without densifications manifest larger survival level as compared to those proceed after densification. The isostatic densification results in lower biocompability of the surfaces. The observed drop was from 23% to 11%, 7% to 4%, and 15% to 8% for W1ssc, W2ssc and W3ssc composites, respectively. The worst biocompatible properties were observed for W2ssc sample. The ability of cells to grow on

Mouse osteoblasts behave similarly. The observed increase was from 18% to 24%, 7% to 11%, and 13% to 19% for W1ssc, W2ssc and W3ssc composites, respectively. Analogously as for fibroblasts, the quality of growing conditions for osteoblasts can be order as follows: the worst biocompatible properties were observed for W2ssc composite, then W3ssc, and W1ssc

structures are superimposed with high contrast against a very dark (or black) background. The limits of detection are generally governed by the darkness of the background, and the excitation light is typically several hundred thousand to a million times brighter than the emitted fluorescence. In the presented studies, the fluorescence microscope was used to visualize the actin filaments, which were stained with phalloidin labeled with Alexa Fluor 488 that absorb blue light (λ = 495 nm) and emits green fluorescence (λ = 518 nm). The images were recorded using Olympus 71X microscope equipped with the 100 W mercury lamp, the MWIG2 filter, and digital camera XC 10 (working under CellR program).

### **4.2 Results and discussion**

The visualization of the cell shape has been perform through actin filament staining using phalloidin coupled with Alexa – Fluor 488. Although actin filaments are dispersed within entire cell, they are concentrating mainly in cortex layer beneath the plasma membrane, and therefore they can be used for cell-shape visualization (fig. 18). The cell cultures were performed for single cells grown for 96 hours. The images observed for fibroblasts were better due to the higher amount of actin filaments inside as compared to osteoblats. However, independenlty of the cell type, their shape varied from rounded to the well extended (spindle – like) ones.

a) b)

Fig. 18. Fluorescent images recorded after 96 hours of growth on composites (magnitude 600x), a, b – fibroblasts, c – osteoblasts.

c)

structures are superimposed with high contrast against a very dark (or black) background. The limits of detection are generally governed by the darkness of the background, and the excitation light is typically several hundred thousand to a million times brighter than the emitted fluorescence. In the presented studies, the fluorescence microscope was used to visualize the actin filaments, which were stained with phalloidin labeled with Alexa Fluor 488 that absorb blue light (λ = 495 nm) and emits green fluorescence (λ = 518 nm). The images were recorded using Olympus 71X microscope equipped with the 100 W mercury

The visualization of the cell shape has been perform through actin filament staining using phalloidin coupled with Alexa – Fluor 488. Although actin filaments are dispersed within entire cell, they are concentrating mainly in cortex layer beneath the plasma membrane, and therefore they can be used for cell-shape visualization (fig. 18). The cell cultures were performed for single cells grown for 96 hours. The images observed for fibroblasts were better due to the higher amount of actin filaments inside as compared to osteoblats. However, independenlty of the cell type, their shape varied from rounded to the well

a) b)

c) Fig. 18. Fluorescent images recorded after 96 hours of growth on composites (magnitude

lamp, the MWIG2 filter, and digital camera XC 10 (working under CellR program).

**4.2 Results and discussion** 

extended (spindle – like) ones.

600x), a, b – fibroblasts, c – osteoblasts.

The round cells denotes the cells that starts to detach from the investigated surface. In general, cells spread and grow on rigid substrates if the provided conditions enable them to preserve normal functioning (in such conditions fibroblasts turn into spindle-shaped cells) but they retract, and become round on non-biocompatible ones. The table 6 presents the summary of shapes observed for both fibroblasts and osteoblasts cultured on the studed surfaces W1ssc, W2ssc, and W3ssc.


Table 6. Summary of cellular shapes cultured on the different composite's surfaces.

To estimate the number of cells present on composite surface, the percentage of cells present on a given sample type was calculated as a ratio of cells present on the studied surface divided by the number of cells cultured on a glass coveslip (2500 and 4800 cells on a glass rounded coveslip with diameter of 10 mm for human skin fibroblats and mouse osteoblasts, respectively).

Figure 19 a and b shows the comparison between the percentage of cells attached to composite surfaces (W1ssc, W2ssc, W3ssc) after 96-hour of growth in culture conditions. In case of fibroblasts, the composite surfaces without densifications manifest larger survival level as compared to those proceed after densification. The isostatic densification results in lower biocompability of the surfaces. The observed drop was from 23% to 11%, 7% to 4%, and 15% to 8% for W1ssc, W2ssc and W3ssc composites, respectively. The worst biocompatible properties were observed for W2ssc sample. The ability of cells to grow on W1ssc and W3ssc was comparable.

Mouse osteoblasts behave similarly. The observed increase was from 18% to 24%, 7% to 11%, and 13% to 19% for W1ssc, W2ssc and W3ssc composites, respectively. Analogously as for fibroblasts, the quality of growing conditions for osteoblasts can be order as follows: the worst biocompatible properties were observed for W2ssc composite, then W3ssc, and W1ssc surfaces are placed.

Biocompatible Ceramic – Glass Composite –

Manufacturing and Selected Physical – Mechanical Properties 247

a)

b)

The doubling time was dependent on the substrate properties. For fibroblasts, the obtained values were 120 ± 15 h, 148 ± 18 h, 117 ± 19 h for composite's surfaces prepared without densification (fig. 20 a). The application of densification step introduces the increase of doubling time to 156 ± 16 h, 167 ± 23 h, and 143 ± 17 h, for W1ssc, W2ssc, and W3ssc, respectively. Longer doubling times correlated well with the lower number of cells present on the corresponding composite's surface. Thus, indicating worse growing conditions for

Fig. 20. The percentage of cells (fibroblasts and osteoblasts) present on a surface of

composite surfaces.

b)

Fig. 19. The percentage of cells (fibroblasts and osteoblasts) present on a surface of composite surfaces.

One of the important conditions that should be provided by biocompatible materials is providing the optimal conditions for cell growth, which can be characterized by the doubling time (Maret D., et. al, 2010, Puttini S., et al, 2009). The chosen cells doubled their amount after 130 and 38 hours for fibroblasts and osteoblasts, respectively, when cultured on Petri dish in optimal conditions. Therefore, this parameter was determined to monitor the changes in their growth. The results are presented in fig. 20 a and b.

a)

b)

One of the important conditions that should be provided by biocompatible materials is providing the optimal conditions for cell growth, which can be characterized by the doubling time (Maret D., et. al, 2010, Puttini S., et al, 2009). The chosen cells doubled their amount after 130 and 38 hours for fibroblasts and osteoblasts, respectively, when cultured on Petri dish in optimal conditions. Therefore, this parameter was determined to monitor

Fig. 19. The percentage of cells (fibroblasts and osteoblasts) present on a surface of

the changes in their growth. The results are presented in fig. 20 a and b.

composite surfaces.

Fig. 20. The percentage of cells (fibroblasts and osteoblasts) present on a surface of composite surfaces.

The doubling time was dependent on the substrate properties. For fibroblasts, the obtained values were 120 ± 15 h, 148 ± 18 h, 117 ± 19 h for composite's surfaces prepared without densification (fig. 20 a). The application of densification step introduces the increase of doubling time to 156 ± 16 h, 167 ± 23 h, and 143 ± 17 h, for W1ssc, W2ssc, and W3ssc, respectively. Longer doubling times correlated well with the lower number of cells present on the corresponding composite's surface. Thus, indicating worse growing conditions for

b)

Biocompatible Ceramic – Glass Composite –

agreement number 213717 (NMP4 - SE - 2008 - 213717).

*Biomedical Materials Research*, pp. 1049-1060

*Szkło i Ceramika,* No.57, pp.16 – 20 (in Polish)

invasion. *Neoplasia (New York, NY)* 12, pp. 1066-1080

**6. Acknowledgments** 

2385 – 2394

1069-1078

588 – 590

Poland (in polish)

Poland (in polish)

*Biomaterials Science*, Springer US,

Koszalin, Poland (in Polish)

*Mechanical Engineering*, No.2, pp. 53 – 64

Poland (in Polish)

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