MINISTERIO DE DESARROLLO URBANO

This chapter is mainly concerned with the development of different zinc containing phosphate-based glasses for hard tissue (bone) engineering applications since zinc is well known for its role in bone metabolism and structure (Seo et al., 2010b). This was performed by adding ZnO ions in different amounts to the quaternary glasses in place of CaO ions.

Previous studies have been carried out to analyse various zinc phosphate glass formulations of (P2O5-Na2O-CaO-ZnO). The results of these studies suggested that zinc contained phosphate glass might have poor cytocompatibility because of its fast dissolution rate. This rate was enhanced as ZnO content increased within the glass network (Salih et al., 2007, Abou Neel et al., 2008c).

The results of this study were designed initially to confirm the successful production of glass discs. This was determined through density and XRD results. Density data displayed the positive relation between the increase in glass density and the incorporation of zinc content within glass which is mainly related to the difference between zinc density (7.14 g.cm-3) and calcium density (1.54 g.cm -3). Whereas XRD findings showed the amorphous nature of the prepared glass discs.

The differential thermal analyses data revealed that all the thermal variables had declined with the increasing incorporation of zinc oxide. This could be linked to the difference in bond enthalpies. When zinc oxide bond enthalpy is

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about 284 Kj/ mol is less than that of calcium oxide bond 464 Kj/mol. Hence, less thermal energy is needed to break Zn-O bonds.

Degradation findings were similar to the DTA where adding ZnO to the glass composition has yielded a faster dissolution rate. This could be explained by two points. The first one is the difference of bond enthalpy and the second one may be due to the difference of bond electronegativity. Both of Zn-O and Ca-O bonds have pure ionic bonds (Ghadah S. Alghamdi, 2013). There is clear variation in electronegativity between both of Zn-O and Ca-O; Ca-O seems to be more electronegative (3.44-1=2.44) than that of Zn-O (3.44- 1.65= 1.79) when 3.44, 1.65 and 1 are the electronegativity of O, Zn and Ca respectively. As a consequence, Ca-O bond polarity and bond strength are greater than that of Zn-O which may explain the glass susceptibility to dissolution when more zinc oxides percentage were added. This could affect the whole glass compositional structure and resulted in this pattern of weight loss seen (ZnO0< ZnO5< ZnO10< ZnO15).

Regarding ion release, it was found that adding zinc oxide content in the glass composition might be correlated with the high release of different phosphate anions and this may be due to the weak bond formation in a zinc phosphate glass as explained previously. Concerning cations, Na+ and Zn2+ ion release was comparable with the anion and mass degradation (ZnO0< ZnO5< ZnO10< ZnO15). The only exception was for Ca2+ ions which was released at higher levels in the glass with ZnO 0mol% glass and this result was the opposite to the previous findings, but that can be interpreted as this

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composition has no CaO substitution, and its CaO content is higher than the others. Hence, it may release more calcium ions. pH results were clearly related with the anion results and that could be explained as phosphate ions are released at high levels and more phosphoric acid may be produced that can decrease the pH. All of these findings were consistent with that of Salih

et al (Salih et al., 2007).

In regard to cell studies, the proliferation study was carried out through the direct counting of live and dead cells using confocal microscopy. This was done with the aid of a template. Many previous studies used live and dead fluorescent stain as a subjective tool for cell proliferation estimation. Though, this technique can have visual bias and poor sample presentation. The main aim in this study was to objectively establish the accurate live and dead cells number, and this was achieved by using the template. Only 13 identified squares per each sample were chosen for the cells counting measurements. Although the total area of these 13 squares composed only 7% of the whole disc area, their positions were selected in a way that ensure the full distribution over the whole central area of the disc to prevent repeated readings and give a valid picture of cell distribution over the disc since each of the selected squares was surrounded by non-interpreted squares. Cells number counting was performed only in the empty areas inside the square and avoiding the square borders that were fluorescent.

Seeding technique for cells was performed gently to attain a uniform cell distribution over the whole discs samples. This was performed through the

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preparation of the intended seeding cell density in 50 µl of culture media which was found to be the optimum media volume to cover the whole disc surface area. The main outcomes of this experiment revealed that live cell number was mostly significantly different between the zinc glasses and control sample for all time points, this does not cancel the positive impact of these two compositions on cell proliferation. Conversely, cells number was apparently low for ZnO15 and ZnO10 formulations and were significant when they were compared with the control group having about half the cell number of the control samples. This was further confirmed by the dead cell counting

data which was linked to zinc oxide content

(ZnO15>ZnO10>ZnO5>ZnO0>control) which suggested that the glass with higher dissolution rate gave poorer cellular related data. Findings from ZnO15 dead cells numbers showed that they composed about 10% of live cell numbers on the same day for the all times points which signified cytotoxicity (Figures 3.9, 3.10).

Alamar blue assay is a useful tool for cell metabolic activity through its ability to be fluorescently influenced by the main by-products of reduction–oxidation (Redox) reaction because the live metabolic cells tend to produce H+ ions that have the capability to reduce the weakly purple fluorescent Resazurin salt to strongly red fluorescent Resorufin (Lancaster, 1996). The alamar blue findings were concurrent with cell proliferation (live and dead cells counting) as it showed that ZnO15 had very low impact on MG63 metabolic activity and they were again clearly significantly different in comparison to the other glass

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formulations at all time points. For ZnO10 data showed little variation from control samples (TCP) and were slightly different with ZnO10 live and dead cells findings. Though it showed an acceptable metabolic activity with time. The other two formulations (ZnO0 and ZnO5) appeared to have slightly lower levels of effect on metabolic activity compared to the control samples which matched that of live and dead cells data. The general trend of alamar blue metabolic activity was (Control>ZNO0>ZNO5>ZnO10>ZnO15). Although there was clear statistical difference (P<0.05) between the control and ZnO0 and ZnO5, these two compositions had a sign of metabolic activity enhancement that might indicate their benign effect on cellular activity. SEM images emphasized the previous cellular related results; ZnO15 SEM images revealed that there was a decline in cell number with poor cellular interconnection.

All of these findings may be explained as a result of the fast rate of glass dissolution of ZnO15. This fast degradation rate has yielded high zinc ion release that could have a negative impact on cell proliferation by suppression of the expression of mRNa as was found in previous studies (Aina et al., 2007, Kwang Hwan Park et al., 2013).‎Interestingly,‎this‎study’s‎results‎were‎ very comparable with the previous study which reported that incorporation of zinc content >1.2 % wt. can lead to cytotoxic effects on osteoblastic MC3T3- E1 cells (Ito et al., 2000). In the present study, Zn composed about 1% wt. of the total glass weight in ZnO15 samples which may explain the undesirable cellular effects seen with this composition. These results described that null

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hypothesis was rejected in regard to ZnO15 glass compositions as they showed signs of poor cytocompatibility. Whereas as it was not rejected in regard to the other zinc glass formulations (ZnO10, ZnO5) and the free zinc glass formulation (ZnO0) that possessed acceptable biological and cellular properties in comparison to the control groups.

In conclusion, although the development of zinc containing phosphate-based glass was successful, adding zinc oxide at 15mol% of the whole glass may have cytotoxic effects. However, adding zinc oxide in the percentage of 5-10 mol% may show better outcomes when compared to the ZnO15 samples. Further work is needed to be carried on some of the ZnO5 and ZnO10 formulations to fully understand their cellular response. Moreover, development of glass in other features that may be more clinically relevant may be needed to carry out as well.

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Chapter 4 Development of strontium and zinc phosphate-