To validate the gelation onset measurements, the gel point of the optimum
formulation, i.e. 2.5% methylcellulose/8% sodium chloride/0.05% carbon dots, was
also determined using oscillatory sweep measurements. The gelation temperature
can be defined by observation of a frequency-independent value of damping factor
(tanδ) obtained from a multi- frequency plot for a temperature sweep
measurement as shown in Figure 5. The gelation temperature was found to be
around 35 °C which is well within the desired range. Figure 6 and 7 exhibit the
synthesized carbon dots and their TEM images, respectively. The spherical shape of
the particles as well as their nanometric particle size range cab be simply observed.
Cytotoxicity testing of hydrogels is being conducted. Preliminary results show
adequate proliferation of dental pulp stem cells, indicating their non-cytotoxic
nature.
37 °C, as demonstrated by the area where data from multiple frequencies reaches intercepting point. This gelation temperature is well within the desired range.
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 30 31 32 33 34 35 36 37 38
Tan
(d
el
ta)
Temperature (°C)
1.000 Hz 1.259 Hz 1.585 Hz 1.995 Hz 2.512 Hz 3.162 Hz 3.981 Hz 5.012 Hz 6.31 Hz 7.943 Hz Approximate Gelation PointFigure 8 - damping factor as a function of temperature at different frequencies for optimum methylcellulose-sodium chloride mixture; the gelation temperature can be defined as the intersection point of all curves
Table 1 - Data using frequency-independent damping factor mode of analysis for a 2.5% w/v Methylcellulose/8.0% w/v sodium chloride
To evaluate gelation, the effect of the addition of carbon dots to the gelation range, the formulation of methylcellulose and 50μg/mL of carbon dots were prepared and
the formulation was evaluated using the shear viscosity (complex component) model on Kinexus+ rheometer. According the data seen in figure 9, the result was not significantly affected by carbon dot addition and gel-sol transition occurred around 35 °C.
Figure 9 - Gelation temperature utilizing shear viscosity (complex component)(Pa s) model after addition of 50μg/mL carbon dots
0 50 100 150 200 250 300 350 400 0 5 10 15 20 25 30 35 40 45 50 She ar V isc osity (c om ple x c om pone nt) (P a s) Temperature (°C)
Gelation Temperature 8.0% NaCl/2.5% MC/
50μg/mL CD
Cell Culture/Cytotoxicity
Biocompatibility of fabricated methylcellulose-based hydrogel was measured by evaluating cell viability of dental pulpal stem cells.
Results were organized in two different plots: the first plot includes grouping by sample where as the second plot includes grouping by time point (Figures 9 and 10, respectively). Based on obtained data, the relative concentration of added carbon dots (25μg vs. 50μg) did not have a significant effect on DPSC proliferation. From these
figures it can be inferred that during the first two days after seeding the cells all four groups produced similar results, with cell counts being below 1000 cells. At day 5 there is a significant increase in cell proliferation for the control group (no gel added), while at 14 days all groups exhibit comparable levels of proliferation.
Figure 10 - Cell proliferation (grouped by samples) results of dental pulpal stem cells (DPSC's) comparing hydrogel preparation with 25μg/mL CD, 50μg/mL CD,
5% MC/8% NaCl and positive control (no gel)
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 25 MC 50 MC MC Control
Cell Proliferation
D1 D2 D5 D14In contrast to the hydrogel cytotoxicity results, figures 12 and 13 demonstrate that the exposure to calcium hydroxide (regardless of the amount and concentration) was detrimental to survival of dental pulp stem cells when compared to a control group. Once again, results of the calcium hydroxide test have been plotted both by time points and by sample. The cell proliferation counts stayed consistently low for all of the calcium hydroxide samples (approximately around 500 cells) for the entire duration of experiment (14 days). The cell proliferation levels of control group reached highest levels at day 5 and slightly decreased by day 14. This is likely due to reaching 100% confluence and not having any more room to proliferate.
Figure 11 - Cell proliferation (grouped by time interval) results of dental pulpal stem cells (DPSC's) comparing hydrogel preparation with 25μg/mL CD, 50μg/mL
CD, 5% MC/8% NaCl and positive control (no gel)
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 D1 D2 D5 D14
Cell Proliferation
25 MC 50 MC MC Control
Figure 12 - Cell proliferation (grouped by time interval) results of dental pulpal stem cells (DPSC's) comparing calcium hydroxide preparations with .15g, .10g, .05g and positive control (no calcium hydroxide)
Figure 13 - Cell proliferation (grouped by calcium hydroxide amount) results of dental pulpal stem cells (DPSC's) comparing calcium hydroxide preparations with .15g, .10g, .05g and positive control (no calcium hydroxide)
0 200 400 600 800 1000 1200 1400 1600 1800 2000 24h 48h D5 D14
Calcium Hydroxide Cell Proliferation
.15g .10g .05g Control 0 200 400 600 800 1000 1200 1400 1600 1800 2000 .15g .10g .05g ControlCalcium Hydroxide Cell Proliferation
24h 48h D5 D14CHAPTER 5
DISCUSSION
The primary objective of the present study was to design an injectable, thermoreversible hydrogel for potential use in endodontics. This hydrogel was
incorporated with carbon dots, a novel nanoparticle that has not been evaluated for use in dentistry. Secondarily, the biocompatibility of such formulation was to be studied by evaluating the proliferation of dental pulpal stem cells when exposed in direct contact to the hydrogel. This was done in comparison to exposure of cells to Ultracal XS, a commonly used inter-appointment endodontic calcium hydroxide medicament. The ultimate goal is to open the possibility of using such novel materials in regenerative endodontic procedures.
The proposed hypothesis that the formulated hydrogel with carbon dots allows dental pulpal stem cells to successfully proliferate was supported by the experimental findings. The ability to safely utilize this material within the prepared canal space of human teeth opens the possibility to multiple future research projects.
A thermo-reversible hydrogel was fabricated using carboxy-methylcellulose powder dissolved in de-ionized water. The effect of addition of various salts to the hydrogel and its effect on gelation temperatures were studied through use of a rheometer. The goal was to fabricate an injectable hydrogel that would initially be maintained in a liquid (sol) state and shift to a more solid (gel) state once it reaches approximate body temperature. From a clinical perspective, creation of such material would allow the clinician to directly inject the hydrogel into a physiologic space (e.g. pulp canal space)
which would then solidify upon reaching physiologic body temperature and would not be washed away. This was accomplished by using a concentration of 8.0% w/v sodium chloride and 2.5% w/v methylcellulose as confirmed by analyzing the complex shear viscosity as a function of temperature.
Carbon dots, a subclass of graphene quantum dots, were prepared using ammonium citrate precursors and functionalized by spermidine molecules for surface added amine groups. In theory, such amine groups would contribute to the antibacterial function of the material. However, within the scope of this study, the antibacterial property of formulated carbon dots was not investigated. Once prepared, the carbon dots were added to the hydrogel formulation and the gelation temperature of the resulting material was once again confirmed using rheometry.
To test the relative biocompatibility of the resulting hydrogel after the addition of carbon dots, a series of experimental plates were set up with viable dental pulpal stem cells. Hydrogels with varying carbon dot concentrations and calcium hydroxide were added to the experimental wells and cell survival was measured and compared between the samples at varying time intervals.
The formulated hydrogel can potentially be used in regenerative endodontics as a scaffold material. In this study, carbon dot nanoparticles were added to the hydrogel. Their use in context of endodontic therapy has not been investigated. A multitude of biomaterials can be introduced and studied due to ability to characterize and functionalize carbon dot nanoparticles. For purposes of endodontic studies, where disinfection of infected root canal system is paramount, antibiotic conjugates would be extremely beneficial to treatment of pulpal pathology. In the context of regenerative endodontics,
the ability to add crucial tissue engineering components (growth factors, stem cells, etc.) to a formulated hydrogel would potentially make the clinical procedure more predictable and not rely on recruitment of autologous cells from the patient/host. Current
regenerative endodontic treatment protocol relies on intracanal irrigants and medicaments to disinfect the canal space, followed by introduction of growth factors, scaffold and stem cells. The procedure is typically performed in two or more visits with effort to promote further root formation and maintaining function in young dentition. Since the procedure is typically performed in a pediatric population where behavior management and patient anxiety are a concern, a single-visit protocol would be advantageous for the patient’s experience. It is the hope that this current study provides a stepping stone towards the development of a more ideal material which would provide a safely administered disinfecting effect while also serving as a scaffold and carrier of tissue engineering components.
As is the case with many studies, this present study has multiple limitations and leaves many unanswered questions. Despite shedding light on the effect of fabricated hydrogel on survival and proliferation of dental pulpal stem cells, many other cells would need to be tested to further understand these effects and make the material applicable for use in regenerative endodontic procedures. These cells include PDL cells and stem cells of apical papilla, among others. The next obvious question that needs to be answered is how the proposed hydrogel would interact with endodontic bacteria and the bacterial byproducts and virulence factors. As mentioned previously, the hydrogel can, theoretically, be characterized with antimicrobial products and medicaments. To this day, certain endodontic pathogens remain notoriously resistant to utilized medicaments
and irrigants, largely due to their biofilm organization. Therefore, investigation of new materials is encouraged.
Another important aspect to consider is interaction between hydrogel and dentinal surface. The eroding nature of both sodium hypochlorite and EDTA have been
extensively studied and evaluated; the evaluation of hydrogel in this respect is imperative to evaluation of safety of this material to study the weakening effects it may have on natural tooth structure.
Methylcellulose was selected for the present study due to its abundance, ease of fabrication and favorable gelation properties. However, many other materials are available and can be investigated for fabrication of injectable hydrogels, each with their unique chemical and physical properties, advantages and disadvantages. Additionally, multiple ways exist for fabrication of graphene carbon dots with the possibility to creating nanoparticles of different sizes. Such variability should be considered in terms to addition to hydrogels, antibacterial properties and biocompatibility.
CHAPTER 6
CONCLUSION
Within the constraints and limitations of this study, the use of cellulose-based hydrogels and carbon dot nanoparticles should be considered as a biocompatible material when exposed to dental pulpal stem cells. It is the hope of authors and researchers involved in this project that these findings pave the road to further investigation and provide a stepping stone towards potential use of injectable hydrogels in dentistry and, more specifically, in endodontics.
From this study, the following conclusions can be made:
1. An injectable hydrogel with gelation range within a physiologic range can be fabricated by using a gel solution of 2.5% w/v carboxy-methylcellulose and 8.0% w/v sodium chloride.
2. The methylcellulose/sodium chloride/50μg/mL carbon dots hydrogel allows for
adequate proliferation of dental pulpal stem cells when compared to calcium hydroxide product Ultracal XS.
3. The finalized hydrogel should be evaluated by further studies in an effort to create a new material for use in regenerative endodontics.
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