Evaluation of cell viability of Human Dental Pulp Stem Cells in Two dimensional and Three dimensional Fibrin Glue Scaffold.

CONTEXT: Regenerative endodontics uses the concept of tissue engineering to restore the diseased immature tooth, allowing for continued development of the root to a healthy state. For regenerative endodontics, the use of human dental pulp stem cells (HDPSCs) with appropriate scaffolds and growth factors is imperative. AIMS: The aim of the study was to evaluate the human dental pulp cell viability in two-dimensional (2D) and 3D fibrin glue scaffold to be used in regenerative endodontics. SUBJECTS AND METHODS: Regenerative potential of HDPSCs was comparatively assessed usings 2D and 3D fibrin glue scaffold. 3D scaffold was made with different concentrations of fibrinogen. Cell morphology was studied under inverted phase-contrast microscopy, and cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at various time intervals. STATISTICAL ANALYSIS USED: Collected data underwent a two-way ANOVA test. The P value of the study was kept 0.05 according to the sample size.
RESULTS: Study revealed a significant increase in the proliferation of HDPSCs in fibrin glue precoated wells of 2D fibrin glue compared to preseeded cells on day 1 and day 3. The concentration of fibrinogen has a major role in cell viability in 3D fibrin glue scaffold. Homing of HDPSCs in the 3D scaffold improves with time.
CONCLUSIONS: This study concludes that the concentration of fibrin glue has a significant role in HDPSC Viability in 3D scaffold. Copyright:

INTRODUCTION

Regenerative endodontics has opened a gateway for a new era of endodontic treatment. Revascularization is widely adopted therapy as of now for the treatment of immature pulpless teeth.[1] To overcome some of the difficulties associated with revascularization, regenerative therapies using dental pulp stem cells (DPSCs) were suggested. Regenerative endodontics is a branch that comprises of adult stem cells to grow and differentiate, growth factors to induce cellular proliferation, and scaffolds for mechanical and biological support to the proliferating cells.[2]

The potential efficiency of stem cell delivery and differentiation may be enhanced with the help of tissue-engineered scaffolds. Delivery of dental stem cells can potentially be supported by scaffolds that provide both mechanical and molecular cues for differentiation.[34] Scaffolds can be natural as well as synthetic based on their process of formation. Both natural and synthetic scaffolds have their merits and demerits. Natural polymers such as collagen, fibrin, silk, alginate, agarose, chitosan, and hyaluronan are biologically active and typically promote excellent cell adhesion and growth. Synthetic scaffolds such as polystyrene, poly-l-lactic acid, polyglycolic acid, and poly-dl-lactic-co-glycolic acid have shown much success as they can be fabricated with a tailored architecture, but they have drawbacks including the risk of rejection due to reduced bioactivity.[5]

Commercially available fibrin glue has been proposed to be a tissue engineering scaffold. It has been widely used as a bioadhesive in surgeries for hemostasis, wound closure, and a sealant.[6789] Fibrin glue has also been successfully applied in cardiovascular, neural, ocular, and thoracic surgeries. In comparison to other polymeric materials, fibrin gel presents many advantages, such as a controllable degradation rate which matches those of tissue regeneration, nontoxic degradation products, and excellent biocompatibility. Furthermore, the morphology, mechanical properties, and stability of fibrin gel could be tuned by controlling the precursor concentration and ionic strength.[10] Its proposed use in DPSC tissue engineering is yet to be explored. Furthermore, it has been described in the literature that fibrin can serve as two-dimensional (2D) as well as 3D scaffold.[11] Changing the concentration of fibrinogen can even change the property of fibrin scaffold resulting in change in cell viability.

Hence, the purpose of the study was to evaluate human dental pulp stem cell (HDPSC) viability in 2D and 3D fibrin glue scaffold to be used in regenerative endodontics. Furthermore, HDPSC viability was tested in different concentration of fibrinogen solution to optimize fabrication and gelation condition of 3D scaffold which supports regeneration ability.

SUBJECTS AND METHODS

All the experimental studies were carried out in a Biosafety Level II equipped Cell Culture Laboratory.

Initial handling of cells

HDPSCs and culture medium were purchased from Himedia Laboratories Pvt Ltd. DPSCs are positive for CD-95 and CD-105 which are HDPSC specific positive markers and negative for CD-34 and CD-45 which are HDPSC specific negative markers. The flask was then incubated in CO2 incubator at 37°C and 5% CO2 till confluency was reached.

HDPSCs need complete medium containing all nutritive elements for growth. For the preparation of complete growth medium, sterile growth medium compatible with stem cells and recommended by manufacturer was measured 50 ml and taken in a sterile flask. 5.55 mL of fetal bovine serum and 0.55 mL antibiotic-antimycotic solution which was passed through 0.22 μ sterile filter was then added to the flask.

The medium of the cells was changed every 24 h. Once the cells had reached 70%–80% confluency, they were used in the experiment.

Preparing two-dimensional and three-dimensional fibrin glue scaffold

The fibrin glue kit (Tissel Lyo, Baxter International Inc.) contains four bottles of fibrinogen (human), thrombin (human), aprotinin (synthetic), and calcium chloride. Aprotinin is mixed with fibrinogen solution till no undissolved particles are visible. Similarly, calcium chloride is mixed with thrombin solution to obtain a homogenous mixture.

HDPSCs were seeded at a density of 3000 cells per well with 300 μL culture medium. Cells with media served as positive control [Figure 1].

Figure 1

Spindle-shaped human dental pulp stem cells. Blue arrow depicting pulpal stem cells. Red arrow depicting cellular matrix

For 2D scaffolds, two sets of experiments were planned. In one set, cells were preseeded. Then, 25 μL of fibrinogen and 25 μL of thrombin were added above it to form fibrin glue. In another set, fibrin glue was initially made in the well plates, and then, cells were seeded on top of it. This set was termed as fibrin glue precoated set.

For 3D scaffolds, 25 μL of thrombin was added, and then, cells with culture medium were added and gently agitated to mix them well. Fibrinogen solution was then diluted to three concentrations (12.5%, 25%, and 50%) by mixing with phosphate-buffered saline (PBS). After that, in different well plates, different concentration of reconstituted fibrinogen solution was added for the glue to form with cells trapped in the fibrin scaffold. Cell morphology was seen at various time intervals under an inverted phase-contrast microscope [Figure 2].

Figure 2

(a) Cells seeded on fibrin glue precoated wells. (b) Fibrin glue treatment on preseeded cells (c) human dental pulp stem cell in three-dimensional scaffold with 12.5% fibrinogen concentration (d) human dental pulp stem cell in three-dimensional scaffold with 25% fibrinogen concentration (e) human dental pulp stem cell in three-dimensional scaffold with 50% fibrinogen concentration. Red arrow depicting human dental pulp stem cells in different scaffolds

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is done to check the proliferation of cells under various conditions. It is a 3-day protocol. The experiment was done in triplicates. On day 1, cells were incubated in the well plate for 24 h so that cells form half-confluent mono-layer and were examined under microscope to make sure that cells were evenly distributed in the well plate. After 24 h incubation, the culture medium was aspirated and in each well, test materials were added and cells were incubated for 24 h at 37°C and 5% CO2. On day 3, the cells were observed under an inverted light microscope to observe their morphology. After that, the entire treatment medium was aspirated and per each well, 4 μL of MTT solution was added in each test well. The plates were incubated for 5 h at 37°C and 5% CO2. After that, MTT solution was removed, and 100 μL dimethyl sulphoxide was added. The plate was swayed and subsequently transferred to plate reader and absorbance was read at 570 nm (Reference was 650 nm). Viability of cells was calculated using the following formula:

where OD (570) a = Measured optical density of test material

OD (570) b = Measured optical density of blank

The readings of MTT assay for the fibrin glue scaffold after 24 h and 72 h interval.

Two-way ANOVA statistical analysis was applied to the data. P value of the study was kept 0.05 according to the sample size.

RESULTS

Statistically, a significant difference was present between fibrin glue treatment on preseeded cells and cells seeded on fibrin glue precoated wells of 2D fibrin glue groups on days 1 and 3 [Table 1 and Graph 1]. Cells seeded on fibrin glue precoated group showed more cell viability than the one with fibrin glue treatment on preseeded cells. Cells seeded in thrombin with 25% fibrinogen showed maximum cell viability on days 1 and 3 in the 3D scaffold group [Table 2 and Graph 2].

Table 1

Cell viability wise distribution in two-dimensional fibrin scaffolds on day 1 and 3

Concentrations	Cell viability (%)		t	P
	Day 1	Day 3
Fibrin glue treatment on preseeded cells	127.00	86.24	2.230	≤0.05 (S)
Cells seeded on fibrin glue precoated wells	241.50	285.75

S: Significant

Graph 1

Cell viability wise distribution in three-dimensional fibrin scaffolds on days 1 and 3

Table 2

Cell viability wise distribution in three-dimensional fibrin scaffolds on days 1 and 3

Concentrations	Cell viability (%)		Z	P
	Day 1: NS	Day 3:S
12.5% fibrinogen	262.00	147.25	1.483	>0.05
25% fibrinogen	347.00	310.25
50% fibrinogen	243.00	268.50

NS: Not significant, S: Significant

Graph 2

Cell viability wise distribution in two-dimensional fibrin scaffolds on days 1 and 3

DISCUSSION

Revascularization therapies have been proved for the treatment of teeth with immature apex with pulp necrosis.[1213] However, due to unpredictability of apical stem cells and instability of induced blood clot, there has been a new “paradigm shift” toward regenerative therapy. HDPSCs in a biocompatible scaffold with growth factors has proved to achieve predictable regeneration. In recent years, the application of fibrin gel scaffold in tissue engineering has become more common. In comparison to the natural polymeric materials, fibrin gel presents many advantages, such as controllable degradation rate which matches those of tissue regeneration, which can help in regeneration of human dental pulp.

New bio-polymer fibrin glue has four components: Thrombin, fibrinogen, aprotinin as well as calcium chloride. Although, formed synthetically, i.e., outside the human body, it mimics the last step of the blood coagulation cascade and results in a clot of fibrin. Fibrino-peptides are removed from fibrinogen by thrombin.[14] Because of these changes in conformational structure and the exposure of polymerization sites, fibrin monomers self-assemble into insoluble fibrin gel.[15] The fibrin clot adheres to the tissue to prevent the leakage of body fluid and provides cell-binding sites for cell attachment, migration, and proliferation to promote tissue regeneration.[16]

Statistically, a significant difference was present between both the 2D fibrin glue groups on days 1 and 3. Fibrin glue precoated group with cells seeded on it showed more cell viability. The cell viability decreased in the group where fibrin glue treatment was done on preseeded cells. That is because the growth medium which was added had to pass through fibrin glue to reach cells. Even though fibrin glue is porous, its viscosity may have reduced permeability of nutrients, growth factors, and respiratory gases to reach the cells adequately. No studies have been reported which has studied the porosity of fibrin glue for medium to pass and reach the cells.

In this study, reconstituted fibrinogen solution was diluted to three concentrations by mixing with PBS, namely 12.5%, 25%, and 50%. This was done in accordance with the statement given by Zhao et al. that the polymerization of fibrin allows the control of gelation times and network architecture by modifying the thrombin and fibrinogen concentration.[17] In a previous study, Catelas et al. also emphasized the effect of fibrinogen concentration on the release of growth factors while discarding the effect of thrombin concentrations. For this reason, in the present study, the concentration of thrombin was fixed in all groups, and the optimum concentration of fibrinogen was determined based on cell proliferation.[18]

Statistically, a significant difference was not present among various concentrations of 3D fibrin glue group on the 1st day, but it was significant on the 3rd day proving that cell homing on 3D fibrin scaffolds is better with the time interval. Highest cell viability on the 1st as well as 3rd day makes 25% fibrinogen the most favorable concentration for cells to stay viable and proliferate. This is supported by previous studies which indicate that the best proliferation of HDPSC occurs when 25% fibrinogen is used in contrast to lower and higher concentrations of fibrinogen.[19] Lower concentration (4 mg/mL) as opposed to higher concentrations (8, 40 mg/mL) of fibrinogen resulted in more adipocyte differentiation (P < 0.05). This finding was supported by a second publication[20] that found that equine mesenchymal stem cell migration into fibrin hydrogels had a much greater magnitude in both autologous and commercial fibrin sealant with lower fibrinogen precipitate solution percentage (25% superior to 50% and 75%).[21] A paper evaluated the long-term (day 56) therapeutic protein expression of differentiated adipose cells in vitro using 3D fibrin sealant scaffolds and found that fibrinogen concentration was important determinant.[22]

Cell viability of the cells suspended in 12.5% fibrinogen decreased on the next day because probably very low concentration of fibrin glue will not possibly provide optimum growth factors and mechanical properties for cells to grow and survive as supported by previous studies.[17] Cell viability of the cells suspended in 50% fibrinogen was lower than that of 25%, probably because too dense fibrin network would provide too compact environment for cells to proliferate and stay viable. Cell viability increased on the next day which also proves that because of too dense network, growth medium would require adequate time to reach cells for them to proliferate. 3D scaffold also mimics the in vivo regeneration pattern. Overall cell viability decreased on 3rd day in 12.5% and 25% group suggesting that cells after a phase of proliferation, enters differentiation stage in accordance with the growth medium.

There was no statistical difference between mean cell viability between 2D and 3D scaffold on both days. However, viability was higher in 3D scaffold with 25% fibrinogen concentration (347 ± 72.12) than in 2D scaffold (285.75 ± 6.01). This is in accordance with a previous study by Hakkinen et al. which found that fibroblasts migrate at least 1.3 times faster in 3D culture compared with their corresponding 2D culture in collagen, fibrin, and cell-derived matrix.[23]

Even with all the advantages of fibrin glue, it also has limitations such as the chance of potential disease transmission; shrinkage of gel, antibody reaction to synthetic aprotinin which still have to be addressed for the wide adoption of fibrin glue in tissue engineering.

CONCLUSIONS

For the first time, our study reports the successful application of fibrin glue scaffolds for regeneration of HDPSCs in 2D as well as 3D scaffold. Within the limitations of this study, it can be proved that the concentration of fibrinogen has a role on the scaffold microstructure which affects the cell viability.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.