Degradation of zinc containing phosphate-based glass as a material for orthopedic tissue engineering
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Phosphate-based glasses have been examined in many studies as a potential biomaterial for bone repair because of its degradation properties, which can be controlled and allow the release of various elements to promote osteogenic tissue growth. However most of these experiments studied either tertiary or quaternary glass systems. This study investigated a qinternary system that included titanium dioxide for degradation rate control and zinc that is considered to have a role in bone formation. Zinc and titanium phosphate glass discs of different compositions were melt synthesized and samples of each composition was tested for different physical, chemical and biological characteristics via density measurement, X-ray diffraction, differential thermal analysis, mass loss, ion release, scanning electron microscopy, biocompatibility studies via live/dead assays at three time points (day 1, 4, and 7). The results showed that the glass was amorphous and that the all thermal variables decreased as zinc oxide amount raised, mass loss as well as ion release increased as zinc oxide increased, and the maximum rise was with ZnO15. The cellular studies showed that all the formulation showed similar cytocompatibility properties with MG63 except ZnO15, which displayed cytotoxic properties and this was confirmed also by the scanning electron microscope images. In conclusion, replacing calcium oxide with zinc oxide in proportion less than 10 % can have a positive effect on bone forming cells.
1 1 Introduction
Since the first synthesis of Bioglass® by L.L.Hench in 1971, various glass compositions have been developed to be examined for their suitability as biomaterials for clinical application . The concept behind bioactive glass was their chemical reactivity and ability to form hydroxyl crystalline apatite on the surface of the implanted material . Silicate-based glass has been used widely for hard and soft tissue repair and many studies were done to explore the use of silicate based glass for bone tissue engineering purposes and found the ability of the silicate glass scaffolds to enhance the cellular attachment and enhance both MC3T3-E1 and osteoblast cell proliferation [3, 4]. However, whilst melt derived Bioglass undergoes some initial degradation, it is relatively insoluble [5, 6], which worked as a driver to develop phosphate glass with its compositionally dependant degradation rate. In addition the components making up a phosphate-based glass may be biologically beneficial to the human body [3, 4]. The initial studies were concerned mainly with simple ternary compositions composed of phosphate, calcium oxide, and sodium oxide and that the calcium played a major role in controlling degradation, as it leads to the formation of non- bridging oxygen (P–O–Ca–O–P) bonds that lead to structural and thermal stability of the developed glass, concluding that the increase in calcium oxide percentages may reduce the degradation rate and enhance the glass chemical durability [5, 6]. However, this was followed by many studies that investigated the addition of other various elements that can play a potential role in hard tissue growth as well as enhancing glass properties such as zinc and titanium.
Zinc is not only an essential element for cell growth, proliferation, and differentiation, but it has an important role in DNA replication, growth hormones, growth factors, and enzyme production . The human skeleton is usually considered the main reservoir for zinc, which is mainly stored in the osteoid layer [8, 9] It also has an influential role on bone growth and mineralization through its ability to activate aminoacyl-tRNA synthetase in osteoblastic cells, as well as preventing resorption processes due to its inhibitory action on osteoclasts [10, 11]. It was also found that zinc containing bioactive glass has the potential property to form hydroxy carbonate apatite layers on their surface after soaking in simulating body fluid . Zinc phosphate-based glass was originally developed to examine its physical and biological effect on bioactive glass and was shown to enhance osteoblast like cells (HOB) cell proliferation and cellular attachment when it was added to ternary phosphate glass at different oxide mole percentages which did not exceed 5 mol % of glass composition . Controversially, it displayed poor biocompatibility on MG63 when it was added to pyrophosphate glass in percentages between 10–20 % as this amount of addition may lead to increased degradation and decline in glass stability [13, 14]. Other work concerned with studying the replacement of calcium with zinc in silicate based glasses in different ratios (0.25, 0.5, 1, 1.5, 2.5, 5) mol % and revealed that zinc incorporation resulted in a lower degradation rate and enhance glass durability, which does not show significant results on cellular physiological activities . The addition of zinc containing silicate glass to biphasic calcium phosphates, which is composed of hydroxyapatite and β-tri calcium phosphate showed a positive impact on rat calvarium-derived osteoblasts cell proliferation and behavior .
Many studies have been performed to investigate the effect of titanium addition to phosphate glass, and it was found that titanium dioxide incorporation can aid in glass stability and decrease the degradation rate and ion release [17, 18]. It was found that an optimal percentage of titanium dioxide is around 5 mol %, which has shown a biocompatible effect on MG63 cell and the addition of titanium dioxide above that amount does not have a clear and significant effect on glass degradation. The reason behind this is that titanium may tend to make resistant covalent P–O–Ti bonds instead of P–O–P that are more vulnerable to hydration [19, 20], but because of the high valency of titanium, there is a finite number of these bonds that can form.
In the current study, different compositions of quaternary and quinternary titanium stabilized zinc phosphate-based glasses were developed by the melt quenching technique , with the titanium dioxide content fixed at 5 mol % in all the compositions and zinc oxide was varied (0, 5, 10, and 15 mol %) in place of calcium oxide content to investigate the impact of adding zinc oxide on the physical and biocompatibility properties of the glass and whether the effect of titanium dioxide incorporation will decrease the degradation rate to offset the increase seen when adding zinc oxide and therefore overcome the deleterious effects of zinc incorporation that was seen in the previous study .
2 2 Materials and methods
2.1 2.1 Glass preparation
Zinc phosphate glass composition
Zinc phosphate glass composition
Glass name and composition
ZnO 0 %
ZnO 5 %
ZnO 10 %
ZnO 15 %
2.2 2.2 Materials characterization
2.2.1 2.2.1 Density determination
2.2.2 2.2.2 Thermal analysis
Differential thermal analysis was performed for the four compositions by using powder of each glass disc.
Three thermal parameters were measured: (1) T g the glass transition thermal temperature, (2) T c the glass crystallization temperature and (3) T m the melting temperature. A Setaram differential thermal analyzer was used in this experiment using a nitrogen environment and heating temperature extended from room temperature to 1400 °C at 20 °C min−1.
2.2.3 2.2.3 Material degradation
Triplicates of each composition were initially weighed then stored in plastic vials (Sterilin tube) with 25 mm pre adjusted pH deionized water to pH7 by (HCl or NH4OH) and incubated at 37 °C. After 1, 4, 7, and 14 days, the solution was removed and kept for ion release measurements. While the glass discs were dried and weighed at each time point to determine mass loss. Following weighing, the discs were stored again in new fresh deionized water solution.
2.2.4 2.2.4 Ion release measurements
Ion release measurement was performed for each stored sample from the degradation study, this was done for the anions (PO4 3−, P2O7 4−, P3O9 3−, P3O10 5−) and the cations (Na+, Ca2+) and the transition metal (Zn2+) by using the ion chromatography systems (ICS1000, ICS 2500, Dionex, Thermo scientific, Hemel Hempstead, UK).
For the cation measurements all the samples were filtered prior to measurement to remove the anions (OnGuard II, Dionex).
2.2.5 2.2.5 Cell studies
Initially, preparation of human osteoblast-like osteosarcoma cell line (MG-63, European Collection of Cell Cultures, Porton Down, UK) was done using standard conditions (37 °C, 95 % air, 5 % CO2, 95 % relative humidity) in Dulbecco’s modified Eagle medium (DMEM, Gibco, Life Technologies, Paisley, UK) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin/streptomycin (PAA Laboratories, GE Healthcare, Chalfont St. Giles, UK).
Metabolic activity of cells
Again triplicate discs of each composition were sterilized as previously mentioned, before being transferred to 24 well plates on which cells were seeded at a density of 10,000 cells per disc in 1 ml of culture media followed by incubation at 37 °C in an atmosphere of 5 % CO2 for 7 days. Cell culture medium was replaced at 3 day periods and the aspirated culture was kept in another 24 cell culture plate. cytocompatibility assay was done at three times points (days 1, 4, and 7) in which a 10 % solution of water soluble tetrazolium salt-8 (alamar blue, ABD Serotec) was added to the aspirated culture then stored in the incubator for four hours at 5 % CO2. Alamar blue is a cell viability assay reagent which contains the cell permeable, non-toxic, and weakly fluorescent blue indicator dye called Resazurin, which is used as an oxidation-reduction (Redox) indicator that undergoes colorimetric change in response to cellular metabolic reduction. The reduced form Resorufin is pink and highly fluorescent, and the fluorescence produced is proportional to the metabolic activity of cells and was detected at 570 nm by a fluorimeter (Infinite M200, Tecan, Männedorf, Switzerland).
Imaging of cells
Scanning electron microscopy (SEM) images were done at the same time points for each sample which were fixed initially in 3 % glutaraldehyde followed by dehydration through a graded ethanol (50, 70, 90, 100 %) then drying by hexamethyldisilazane (Aldrich, UK).
2.3 2.3 Statistical analysis
Cell work data for both cell counting and metabolic activity studies were statistically assessed by hierarchal ANOVA to find the statistical significance between individual groups and the differences were considered statistically significant at p < 0.05.
3 3 Results
3.1 3.1 Density measurement
Fig. 1 shows glass density (g cm−3) in relation to different replacements of calcium oxides with zinc oxides and revealed that density cumulate as the content of incorporated metal oxide increase, with densities ranging from 2.6 ± 0.004 g cm−3 for zinc free quaternary glass to stand at 3.16 ± 0.03 g cm−3 for Zn15.
3.2 3.2 Thermal analysis
3.3 3.3 Degradation study
3.4 3.4 Ion release measurement
3.5 3.5 Cytocompatibility and metabolic activity
Conversely and as expected, dead cells follow the opposite pattern to that of live cells, in that dead cell numbers rise as zinc percentage increases and was maximum for ZnO15 for all the days (Fig. 6b). By day 1, dead cells number were about 160 ~ 400 cells/disc just half that of ZnO15 that was about 800 cells/disc, which was significantly higher than the other compositions. By day 4, dead cells number increased by five times for all glass types and was again significantly different in ZnO15 to the other types (p < 0.05). By day 7 dead cells number continued to increase especially in the zinc containing glass with maximum number of non-viable cells was in ZnO15 samples which was three times that of control samples.
The continuity in reduction percentage persists to day 7, which was about 42 ± 3.3 % for control and was not as high as that for ZnO0. Despite the presence of significant difference between ZnO5 and Zn10 with the control, the mean difference in reduction was little low from the control about (6.5–9 %). Interestingly, reduction rate for ZnO15 showed a clear decline in day 7 to end at 2.5 ± 0.7 % slightly higher than that at day 1 and displaying higher significant difference with the whole batches.
4 4 Discussion
The present study concerned with the development of different phosphate-based glasses for hard tissue (bone) engineering applications, and this was done by substituting the calcium oxide with zinc oxide, since zinc is known to play important roles in the bone metabolism and development . Previous studies have been carried out to assess different zinc phosphate glass properties, these studies assessed (P2O5–Na2O–CaO–ZnO) phosphate glass and showed that these compositions suffered from poor cytocompatibility due to their rapid degradation rate and this rate increased with increasing zinc addition [13, 14]. Other studies were performed with the incorporation of various amount of titanium dioxide to the tertiary based glass to improve its cytocompatibility and enhance its durability. titanium dioxide was added at 3 mol %, 5 mol %, 7 mol % and revealed that as titanium dioxide increased, the degradation rate and ion release rate decreased. Also the 5 mol % titanium dioxide containing glass showed the most favorable cytocompatibility results in comparison to the titanium dioxide free control samples  and this was the main rationale for the selection of this titanium dioxide content in the present study.
The thermal properties reduced with incorporation of zinc oxide and this tends to be as a result of the fact that zinc oxide bond enthalpy is about 284 Kj/mol, which is less than that of calcium oxide bond 464 Kj/mol as a result of this less energy is needed to break the zinc oxide bond.
Usually the main factors that control degradation of material are the atom size, bond length and electronegativity of bond. Although both of Zn–O and Ca–O bonds have pure ionic bonds [23, 24] as the difference in electronegativity of both Zn–O and Ca–O are more than 2, 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 has more bond polarity and bond strength of Zn–O, which may explain the glass susceptibility to degradation as zinc percentage increase that could affect the whole glass compositional structure and resulted in this pattern of weight loss (ZnO0 < ZnO5 < ZnO10 < ZnO15).
The ion release results were concurrent with the weight loss pattern. It was found that the increase of phosphate anion release increased as the zinc oxide content increased, this may be due to the effect of weak Zn–O–P bond. For the cation release it took the same trend for Na+ and Zn+2 ions, the only exception was for Ca+2 ions, which was released at higher levels in the glass with ZnO 0 mol % glass and this result was the opposite to the other ions, but that can be explained by the fact that as the ZnO content decreased, the CaO content increased. however the general information that was obtained from zinc oxide containing glass ion release was that there was a gradual release in all the ions with time which was similar for the compositions ZnO0, 5, 10 mol %, but the release for the ZnO15 mol % was higher and both findings OD mass loss and ion degradation were consistent with that of Salih et al. .
5 5 Conclusion
The present study showed that adding zinc to the phosphate based glass can be prepared successfully and displayed acceptable outcomes concerning cellular metabolic activities. However the increase in zinc oxide concentration to 15 mol % may have cytotoxic effects, adding zinc oxide in the percentage of 5–10 mol% may show acceptable effects when compared it with the control sample.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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