The degradation of crosslinked KGM hydrogels with increasing concentration of KGM was then observed in aqueous solution after 0-96 hr (Figure 4.10). It was observed that the hydrogels with higher concentrations of KGM (1.25-1.5% (w/v)) started to break up faster compared to hydrogels with lower concentration (0.5-1% (w/v)), suggesting that hydrogels with higher concentrations of KGM, hence with greater amount of OH groups have a greater capacity to absorb more water and higher degree of hydrolysis. In this case, the hydrogel with
184
higher concentration of KGM showed faster disintegration from the inside (bulk degradation). This involves dissolution and degradation of surface layers and also involve with the leaching of unbound or loosely bound components or an uptake of fluids into the hydrogel. The observations on the integrity of the hydrogels are summarized in Table 4.4.
Table 4.3 Qualitative observations of the degradation of crosslinked KGM hydrogels with increasing concentrations of Ce(IV) in aqueous solution after 24, 48 and 96 hr in 12 well plates.
Table 4.4 Qualitative observations of the degradation of crosslinked KGM hydrogels with increasing concentrations of KGM in aqueous solution after 24, 48 and 96 hr in 12 well plates. % Concentration of Ce (x 10-3) (w/v) Time (hr) 0 12 24 48 96
1 Solid Solid Start to break 60-70%
dissolved
Dissolved
1.5 Solid Solid Start to break 60-70%
dissolved
Dissolved
3 Solid Solid Solid Start to break Start to break
6 Solid Solid Solid Solid Start to break
% Concentration
of KGM
Time (hr)
0 12 24 48 96
0.5 Solid Solid Start to break 60-70%
dissolved
Dissolved
1 Solid Solid Start to break 60-70%
dissolved
Dissolved
1.25 Solid Start to
break
Dissolved Dissolved Dissolved
1.5 Solid Start to
break
185
Figure 4.9 Images of the degradation of KGM hydrogels with increasing concentrations of Ce(IV) in aqueous solution (0 to 96 hr).
Figure 4.10 Images of the breakdown of KGM hydrogels with increasing concentrations of KGM in aqueous solution (0 to 96 hr).
186
4.3 Discussion.
Biodegradable KGM hydrogels were successfully synthesized in aqueous conditions using Ce(IV) and KGM at various concentrations. Ce(IV) was used to initiate the hydrogen on the alpha position of hydroxyl group on KGM to form crosslinks and then the concentration of the initiator was increased to create more crosslinking that would improve the hydrogel‘s mechanical and physical properties by strengthening the hydrogel. Hydrogels with higher concentrations of Ce(IV) were able to sustain their shape for more than 96 hr compared to other hydrogels with lower concentrations. For further experiments, 1x10-3% (w/v) Ce(IV) was chosen in the formulation of hydrogels with increasing concentrations of KGM. The hydrogels with higher KGM concentrations (1.25-1.5% (w/v)) degraded faster compared to those with lower concentrations of KGM, indicating that higher concentrations of KGM with more hydroxyl groups were able to absorb more water inside the hydrogel, which then broke the bulk formation from the inside from hydrolysis.
Biocompatibility is an essential issue for biomaterials. It is important to ensure that the hydrogels will not provoke any inflammatory response while their degradation products must be safe to cells. Initially, the effect of hydrogels in indirect contact with skin cells were examined in order to investigate any release of potential toxic products that might be harmful to cells. Indirect contact studies showed that increasing concentrations of Ce(IV) and KGM in crosslinked hydrogels did not stimulate or adversely inhibit the viability of fibroblasts and keratinocytes after 3 days.
The direct contact study of hydrogels with increasing concentrations of Ce(IV) with skin cells showed that all hydrogels inhibited keratinocytes whereas only those with concentrations of 3-6x10-3% (w/v) Ce(IV) were inhibitory to fibroblasts (Figure 4.4). There are two reasons that may contribute to the inhibition of keratinocyte proliferation, i) the expansion and the
187
breaking of the hydrogel that exposed a larger area of KGM onto the cell surface and ii) the presence of Ce(IV) in the hydrogel. The crosslinked KGM hydrogel with 1x10-3% (w/v) Ce(IV) stimulated fibroblast viability after 3 and 5 days almost as equivalent to 10 mg.mL-1 KGM. The Ce(IV) concentration was then used in the formulation of crosslinked hydrogel with increasing concentrations of KGM.
Next, the effect of hydrogels with increasing concentrations of KGM in direct contact with skin cells showed that hydrogels with 0.5-1.25% (w/v) KGM stimulated fibroblast viability while 1.5% (w/v) KGM did not. On the other hand, all hydrogels with 0.5-1.5% (w/v) KGM inhibited keratinocyte viability which was consistent with KGM‘s effects on keratinocyte viability (Shahbuddin, Shahbuddin et al. 2013). The inability of crosslinked hydrogels with 1.5% (w/v) KGM to stimulate fibroblast viability may be attributed to the alteration in KGM hydrogel‘s physico-chemistry from rapid absorption of the medium in the culture well that possibly affects the interaction of cells with materials. From these results, it was observed that 1% (w/v) KGM with 1x10-3% (w/v) Ce(IV) achieved optimum conditions to stimulate fibroblast proliferation compared to other formulations.
The next part in this chapter discusses the characterization of the crosslinked hydrogels with increasing concentrations of Ce(IV) using DSC and FTIR. The DSC thermograms of crosslinked KGM with increasing concentration of Ce(IV) show the trend of increasing of temperature of the endothermic peaks from 80 to 217oC and the increase of the endothermal and exothermal energies (Figure 4.3). The hydrogels with 3x10-3% and 6x10-3% (w/v) Ce(IV) had two distinct peaks of endo and exothermic, which were not observed in the hydrogels with lower Ce(IV) concentrations. The exothermic peaks were due to the increase in the crosslinking in the hydrogels which absorbed more energy in order to immobilize and break KGM polymeric chains. It also presumed that unremoved high concentrations of Ce(IV) in the hydrogels contributed to the factor.
188
The FTIR spectra of crosslinked KGM hydrogels with increasing concentrations of Ce(IV) showed the presence of β-1,4 linked glucosidic and β-1,4 linked mannosidic linkages at 1027-1244 cm-1 (Figure 4.4) which were seen between 1200-900 cm-1 in xylans, chitin, carrageenan and cellulose (Černáa, Barros et al. 2003). The spectra in the region 1200-700 cm-1 also give information about the conformational changes and structures between the hydrogels whereas diminishing spectra bands and differences in relative intensities of the bands at 1700-1500 cm-1, 1300-1100 cm-1 and 900-700 cm-1 were likely due to significant steric reaarangement and interactions in C-OH due to crosslinking (Nikonenko, Buslov et al. 2005).
4.4 Conclusions.
In conclusion, biodegradable, crosslinked KGM hydrogels were successfully synthesized in aqueous medium using Ce(IV) as an initiator and at room temperature. The synthesis and characterization of the hydrogels with increasing concentrations of KGM or Ce(IV) were defined. The results showed that although increasing concentrations of Ce(IV) were able to sustain the hydrogel‘s physical integrity for more than 96 hr, and very inhibitory to both skin cells. The exact mechanism of the hydrogel‘s biological activities on skin cells is unknown, but is likely to be related to the effect of native KGM (A. konjac Koch) as mentioned in the previous chapter. The biodegradibilityof the hydrogels and their ability to stimulate fibroblast viability are very appealing for applications in wound healing.
189