Tri-Lineage Differentiation Potential of Stem Cells from Human Exfoliated Deciduous Teeth - An In-vitro Study

ABSTRACT

Background:

The use of SHED might bring advantages for tissue engineering over the use of stem cells from adult human teeth as SHEDs were reported to have higher proliferation rate and increase cell population doublings as compared with stem cells from permanent teeth.

Objective:

The study’s objective was to assess the growth kinetics and tri-lineage differentiation capacity of stem cells derived from human exfoliated deciduous teeth.

Material and Method:

After being propagated through multiple sequential subcultures, adherent fibroblastic cells in the pulp tissue culture from human exfoliation teeth were evaluated to assess their tripotent differentiation potential into bone, cartilage, and adipose cell lineages.

Result:

The stem cells derived from dental pulp had a comparatively high ability for proliferation and successfully differentiated into chondrocytes and osteocytes while less significant for adipocytes.

Conclusion:

Stem cells from Human Exfoliated Deciduous teeth are mesenchymal stem cells which are multipotent and can serve as a promising incentive for therapeutic and future research purposes.

INTRODUCTION

Mesenchymal stem cells (MSCs) can be obtained from a variety of tissues, such as the umbilical cord, endometrial polyps, menstrual blood, bone marrow, and fat tissue. These cells have been successfully isolated and differentiated from several sources, including bone, fat, amniotic fluid, umbilical cord blood, blood from outside the body, and cartilage. Each of these sources comes with its own set of advantages and disadvantages.[1,2,3,4,5,6,7,8]

There have been reports of MSCs increasing their rate of division and doubling of cells, which could make them a more favorable option for tissue engineering compared to adult human teeth. Moreover, MSCs from human exfoliated deciduous teeth (SHEDs) can be harvested from a tissue that is both easy to access and suitable for young children who have experienced trauma leading to pulp necrosis in their primary teeth. This is particularly relevant to young kids who have injured their permanent teeth before they have fully developed. The aim of the study was to examine the ability of these cells to differentiate into three different cell types and their growth rate.[9,10]

METHODS AND MATERIAL

Thirty removed exfoliated deciduous teeth that had healthy pulp were obtained from children aged 6–14. Cell cultures of mesenchymal stem cells were extracted from the pulp of these teeth. The original cultivation method was used, which involved enzymatic separation using tripsine and colagenase in a medium called DMED-KO. Immediately following extraction, the tooth was treated with colaginase mix or tripsine and placed in a DMEM F12 medium for 5–7 days. Following that period, the migrating pulp cells were tripsinised, filtered into secondary cultures, and cultivated in the same medium.

Cell passaging

New media was swapped out every 48 hours during the study period. During this time, a number of typical small-scale photos of the flasks were captured. Once the cells had reached an 80% growth stage, the old media was discarded, and the cells were washed with DPBS and then separated using 0.25% trypsin. Following this, the cells underwent neutralization and were transferred to a centrifuge tube, where they were rotated at 1800 rpm for 5 minutes. Subsequently, the cells were reintroduced into bigger vessels, with each new batch being grown one step at a time, while a reserve of cells was kept in liquid nitrogen.

Tri-lineage differentiation

Cell lineage induction was carried out once cultures had attained 90% confluence. Adipogenic media (Dexamethasone 1 μM, IBM × 0.5 μM, Indomethacin 100 μM, Insulin 1 μg/mL), osteogenic media (Dexamethasone 10–8 M, Ascorbic acid 50 μg/mL, β-glycerophosphate 10 mM), and the STEMPRO Chondrogenesis Differentiation Kit (Invitrogen) were employed. Cells were preserved for periods ranging from 7–21 days before they were treated with Von Kossa’s stain for osteocytes, Oil Red O for adipocytes, and Alcian Blue stain for chondrocytes. The process of differentiation into specific cell types occurred three times for each line. The STEMPRO Chondrogenesis Differentiation Kit from Invitrogen was used for the transformation of cells into chondrocytes.

Population doubling (PD)

The number of cells was tallied at the conclusion of each segment when they had achieved a confluence of 85%–90% due to SHED expansion. The rate of population growth (PD) was determined by applying the following equation:

PD = [LogN_t-LogN₀]/Log2; N₀ represents the starting amount of cells, while Nt signifies the total number of cells at the end of each stage.

Cumulative population doubling (CPD)

CPD was calculated by summing the population growth factor for every stage and adding it to the growth factor of the previous stage.

CPD = [Log₁₀ (N_t/N₀] *3.33; N₀ stands for the initial count of cells, and Nt is the total count of cells left after every step.

Population doubling time (PDT)

It is a quantification of how quickly a particular cell group within a culture is dividing. The cells were spread evenly across all the wells of a 24-well plate, with each well having an area of 100 μm². The cells in three wells were individually counted at 24, 48, and 72 hours, and then continued to be counted at the same intervals, up until the 192^nd hour (8^th day), with the data being plotted on a graph, with the x-axis representing the number of cells in milliliters (cells/ml) and the y-axis representing time in days.

RESULTS

Human stem cells obtained from deciduous teeth shed by humans were effectively separated and multiplied. The process of turning them into cartilage known as chondrogenesis was identified using Alcian Blue to mark proteoglycans. Similarly, turning these cells into bone was indicated by the marking of a mineral-rich matrix with Alizarin Red at day 21, achieving satisfactory outcomes. However, the conversion of these cells into fat cells—a process known as adipogenic differentiation in human deciduous teeth—showed less success, with only a small number of cells in each microscopic field showing signs of becoming adipocytes. The rate at which these cells doubled decreased over time [Figure 1], but the total number of cell doublings increased [Figure 2]. We managed to grow the cells up to 35 times from the initial to the tenth step [Figure 3]. Nevertheless, the gradual increase in the time it took for the cells to double suggests that these stem cells are aging, leading to a reduction in their ability to regenerate and a decline in tissue health.

Figure 1.

Plot of average population doubling against each passage number.

Figure 2.

Plot of average Population Doubling Time - as cell number (cells/ml) against time (hrs)

Figure 3.

Plot of average population doubling against each passage number.

DISCUSSION

According to recent research, mesenchymal progenitors called SHEDs that were separated from the pulp of human deciduous incisors showed remarkable multipotency, since they could transform into osteoblasts, chondroblasts, and adipocytes. (Miura et al., 2003).[9] This particular study was conducted to assess the potential for tri-lineage differentiation and proliferation of stem cells derived from human exfoliated deciduous teeth, taking into account the previously mentioned facts. Our findings showed that SHEDs have a definite capacity for osteogenesis and chondrogenesis but not for adipogenesis. However, these results are consistent with the findings of Gronthos et al.[11] and Noriaki et al.[12] In a subsequent investigation, Gronthos et al.[13] and a few other writers revealed that dental pulp stem cells might, albeit unintentionally, produce adipocytes.

Because these tissues are not native to the dental pulp cavity or its surrounding tissues, the possibility of osteochondrogenesis and adipogenesis may appear surprising However, the ectomesenchymal origin of the tooth pulp could explain the osteo- and chondrogenic repertoire. During long-term cell cultivation, SHEDs reduce proliferation rates and increase doubling time similar to the study done by Huang et al.[14]

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.