Mesenchymal stem cells from orthodontic premolar teeth

Abstract

Background

Considering the limitations in isolating Bone Marrow Mesenchymal Stem Cells (BMSCs), alternate sources of Mesenchymal Stem Cells (MSCs) are being intensely investigated. This study evaluated dental pulp MSCs (DP-MSCs) isolated from orthodontically extracted premolar teeth from a bone tissue engineering perspective.

Methods

MSCs isolated from premolar teeth pulp were cultured and studied using BMSCs as the control. Flow cytometry analysis was performed for the positive and negative MSC markers. Multilineage differentiation focusing on bone regeneration was evaluated by specific growth induction culturing media and by alkaline phosphatase (ALP) activity. Data were compared by repeated measurement analysis of variance and Student's t-test at a p value <0.05.

Results

Proliferation rate, population doubling time, and colony formation of DP-MSCs were significantly higher (p < 0.001) than BMSCs. More than 85% of DP-MSCs expressed CD44, CD73, CD90, CD105, and CD166. Negative reaction was found for CD11b CD33, CD34, and CD45. Positive reaction was displayed by 7.2% of cells for early MSC marker, Stro-1. Both the cell populations differentiated into adipogenic, osteogenic, and chondrogenic lineages, with adequate ALP expression.

Conclusion

Because DP-MSCs from orthodontic premolars hold a neural crest/ectomesenchymal ancestry, its prudent growth characteristics and multilineage differentiation open up exciting options in craniofacial tissue engineering.

Introduction

Stems cells are foundation cells that are capable of self renewal and differentiation. They form at different stages of mammalian development and are also seen at specific anatomic sites.¹ Accordingly, they are classified as embryonic and adult stem cells, with varying abilities for proliferation and regeneration. Contemporary research in regenerative medicine with cell therapy is predominantly focused toward “adult stem cells” due to the ethical issues, tumor and immune responses of “embryonic” and “induced pluripotent stem” cells.^{2, 3} Adult stem cells are multipotent—more tissue specific with limited differentiation potential. During embryogenesis, they form after germ layer differentiation: neural/skin stem cells from ectoderm, hematopoietic stem cells/Mesenchymal Stem Cells (MSCs) from mesoderm, and most of the organ-specific stem cells from endoderm.⁴ Generally, adult stem cells maintain homeostasis and wound repair.⁵ Stem cell populations are identified on the basis of antigenic surface markers (cluster of differentiation [CD]) and their capacity to differentiate into multilineages. Their proliferation is mediated through transit amplifying genes and progenitor cells, whereas the plasticity or lineage switch is determined by the interplay of external cues, autocrine/paracrine/endocrine molecules, and a multitude of signaling pathways and their downstream regulations.⁵ Among all the adult stem cell types, MSCs have recently generated tremendous interest in stem cell–based therapy and regeneration because of their exceedingly high plasticity.⁶ They are also termed as mesenchymal stromal cells considering their cellular heterogeneity.

Mesenchymal Stem Cells show immune modulation and homing to inflammatory sites, a feature largely determined by its local microenvironment or niche. This makes it useful in immune/non-immune/gene therapies and for tissue repair in injury, trauma, ischemia, burns, radiation, and degenerative diseases.⁶ Scaffold-based delivery of MSCs to critical bone defects is an emerging treatment preference, when repair is not feasible with host bone alone. Scaffolds can render a suitable niche for MSCs for bone regeneration along with bone inductive molecules/signals.⁷ For this, MSCs from bone marrow of long bones, mainly from the iliac crest are currently being used.⁸ However, MSCs comprise only 0.01% of cells in the bone marrow; averaging one MSC per 100 mononuclear cells.⁹ There are other limitations as well for bone marrow–based MSCs: decline in osteogenic potential with age, limited in vitro expansion, and senescence with repeated cell passaging.⁸ Therefore, current thrust is on seeking alternate sources of MSCs from cartilage, synovium, fat, placenta, umbilical cord, tonsil, thymus, and muscle and pulp-periodontal complex in oral cavityfor bone tissue engineering.¹⁰

Dental pulp is a neurovascular tissue from which stem cells were first identified by Gronthos et al¹¹ in 2000 from the third molar teeth. Thereafter, MSCs have been isolated and characterized from different sites in oral cavity such as dental pulp mesenchymal stem cells (DP-MSCs) from human exfoliated deciduous teeth (SHED) and third molar, periodontal ligament stem cells, stem cells from apical papilla, and dental follicle progenitor cells.^{11, 12, 13, 14, 15} These MSCs share a unique ontogeny to “cranial neural crest cells”, which are highly specialized cells that determinedly influence craniofacial development through its subcell populations. Ectomesenchymal cells ventral to the neural tube form bone, cartilage, connective tissue, and dentin whereas non-ectomesenchymal cells which are dorsal to the neural tube give rise to neurons, glia, and melanocytes.¹⁶ It, therefore, is known that MSCs isolated from ectomesenchymal lineages would be the right selection for craniofacial bone tissue engineering. However, none of the aforementioned MSCs reported from pulp-periodontal complex has so far translated into clinical therapeutic use.

In this context, “premolar teeth” extracted for orthodontic treatment, performed for correcting facial/dental malocclusion, are a novel and hopeful source of pulp for MSC isolation. This is because, unlike deciduous/third molar teeth, orthodontic premolars are not affected by caries/periodontal diseases. Because orthodontic treatment is generally performed for adolescent age group children, a high titer of MSCs can be expected from the healthy and hygienic premolar teeth.¹⁰ Isolation of MSCs from premolar tooth pulp therefore offers plausible opportunities in craniofacial bone tissue engineering, stem cell banking, and personal medicine.¹⁷ Furthermore, it is easily and sufficiently available without any ethical issues. Nonetheless, to the best of our knowledge, MSCs from “orthodontically extracted premolar teeth” have not been reported till now. The aim of this study was, therefore, to isolate and characterize mesenchymal adult stem cells from premolar teeth extracted for orthodontic treatment and to compare its growth and multilineage potential with bone marrow–derived MSCs from a bone tissue engineering standpoint.

Materials and methods

Isolation of mesenchymal stem cells from premolar tooth pulp

Premolar teeth were collected from children (aged 14–19 years) undergoing orthodontic treatment at the outpatient department of the institute. Immediately after extraction, teeth were placed in sterile chilled Phosphate-Buffered Saline (PBS) solution with 1% penicillin-streptomycin (Himedia, Mumbai, India) in ice. From these teeth, pulp tissue was removed and enzymatically digested with collagenase type I (3 mg/ml) and dispase II (4 mg/ml) solution (Sigma–Aldrich, St. Louis, MO, USA) for 1 h at 37°C (Fig. 1). Thereafter, the cell suspension was filtered and tested for viability by trypan blue exclusion test.

Fig. 1.

Steps of pulp tissue removal from premolar teeth: (a) exposure of pulp chamber at the cementoenamel-junction, (b) pulp tissue extraction, (c) extracted tissue in phosphate-buffered saline (PBS), and (d) crown and root after removal of pulp tissue.

Dental pulp MSCs (DP-MSCs) were isolated based on their property of adherence to surfaces.¹¹ Cells obtained from the pulp tissue were cultured in Dulbecco's modified Eagle's medium (Sigma Chemical Co. St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco®, Waltham, USA) and 1% antibiotics and incubated at 37°C with 5% CO₂. Non-adherent cells were removed after 3 days, and culture was replenished with fresh growth medium. After obtaining confluent growth, cells were harvested by incubating with 0.25% trypsin-Ethylene Diamine Tetra Acetic Acid (EDTA) (Sigma–Aldrich) at 37°C followed by centrifugation for 5 min at 300 g. Pellet was resuspended in growth media, and cells were subcultured.

Human Bone Marrow Mesenchymal Stem Cells (BMSCs) in vials were procured from PromoCell, Heidelberg, Germany. Passage 3 cells of DP-MSCs and BMSCs were used for all the experiments.

Growth kinetics study

“Cell growth assay” was carried out by seeding MSCs from dental pulp tissue and bone marrow in 6-well plates at 1 × 10⁴ cells/well. Cells were harvested each day till day 9 and counted with hemocytometer. Average number of cells was determined, and the growth curve was plotted.

For assessing “population doubling time” (PDT), DP-MSCs and BMSCs were seeded in 6-well plates at 1 × 10³ cells/well. Cell density at day 14 (T) and initial cell density (T_o) was used for estimating PDT using the formula: [PDT = (T − T_o) lg2/(lgN − lgN_o)] where, T = total culture time, T_o = initial culture time, N = No. of cells at time T, N_o = No. of cells at T_o.¹⁸

The ability to form a colony from a single cell was assessed by “clonogenic assay.” It was performed by seeding both the cell populations in 60-mm plates at 100 cells/plate. After 14 days, cultures were fixed with 4% paraformaldehyde and stained with 3% (v/v) crystal violet (Sigma–Aldrich) for 5 min. Cells were washed using distilled water to remove extra stain, and then umber of colonies was counted. Aggregates of ≥50 cells were scored as colonies.

Immunophenotyping

Phenotype characterization of the isolated DP-MSCs and BMSCs was performed by confirming expression of surface markers with flow cytometry analysis. The positive MSC-specific markers selected for the analysis were Stro-1, CD44, CD73, CD90, CD105, and CD166 and negative markers (hematopoietic markers) were CD11b, CD33, CD34, and CD45. Stock cell suspension (100 μl; 2 × 10⁶ cells of passage 3 to 5 in 1-ml PBS solution) was treated with 10 μl of fluorescent-conjugated antibody and incubated in dark at 4°C for 30 min. Cells were centrifuged at 500 g for 5 min and washed twice with PBS before analyzing with a flow cytometer (BD LSRII, San Diego, CA, USA). Antibodies used were phycoerythrin (PE)-conjugated CD105, CD73 (Santa-Cruz Biotechnologies, CA, USA), CD44 (BD Pharmingen, San Diego, CA, USA), CD11b and CD166 (BioLegend, San Diego, CA, USA) and fluorescein isothiocyanate–conjugated CD90, CD34, CD33, and CD45, and Stro-1 (BD pharmingen, San Diego, CA, US). Isotype control antibodies were used to verify non-specific binding. Data acquisition and analysis were performed using FlowJo software (version 5.5; Tree Star Inc.).

Morphology and contact-dependent growth of MSCs in culture was evaluated by immunofluorescence staining with PE-labeled CD105 antibody on cell monolayer on glass cover slips.

Multilineage differentiation

Cells from passages 3–5 were used to evaluate differentiation potential of DP-MSCs and BMSCs to adipogenic, osteogenic, and chondrogenic lineages. Cells (2.5 × 10⁴) were grown in 60-mm culture plates to reach a subconfluent growth. Then, growth medium was replaced by respective differentiation media. For adipogenesis, 0.5 μM of dexamethasone, 0.5 mM of isobutylmethylxanthine, and 60 μM of indomethacin were added to basal medium. Dexamethasone 10⁻⁸ M, 20 mM of β-glycerophosphate, and 0.2 mM of ascorbic acid were supplemented for osteogenesis. For chondrogenesis, additives were 10% of insulin-transferrin-selenium, 10 μm of ascorbate-2-phosphate, 10⁻⁷ M of dexamethasone, and 10 ng/ml of transforming growth factor beta-1. Negative controls were the cells cultured in basal medium. Cultures were maintained for 21 days in humid atmosphere containing 5% CO₂ at 37°C. Subsequently, cells were stained by Oil red O (0.5% in isopropanol) for intracellular lipid droplets/adipogenic differentiation. Two percent of alizarin red and alcian blue staining were used to assess osteogenic and chondrogenic differentiation, respectively. All reagents were purchased from Sigma–Aldrich.

The osteogenic differentiation ability of both MSCs was further confirmed by estimating alkaline phosphatase (ALP) activity of the cultured cells at day 7, 14, and 21. Colorimetric assay using p-nitrophenyl phosphate was performed after quantitating the protein of cell lysate by Bradford assay.

Statistics

All experiments were carried out in triplicates, and data were represented as mean ± standard deviation. “Repeated day” readings were compared by repeated measure analysis of variance (ANOVA) general linear model, and “one-time” readings were compared using Student's t-test. For all statistical evaluations, a two-tailed probability of value p < 0.05 was considered significant.

Results

Dental pulp stem cells were collected from premolar teeth. Attachment of cells to the culture flask surface was observed after 1–2 days of the primary culture for DP-MSCs and BMSCs. On the 3rd–4th day, DP-MSCs showed typical spindle-shaped fibroblast morphology as shown in Fig. 2 (a and b). Immunofluorescence staining with the highly expressed marker, CD105, verified the spindle morphology and contact-dependant growth of these MSCs as shown in Fig. 2 (c and d).

Fig. 2.

(a and b). Spindle-shaped, fibroblast-like morphology of DP-MSCs at 10× and 20× magnification, respectively; (c and d) Immunofluorescence staining for surface marker CD105 (a) DPSCs and (b) BMSCs. Cell–cell contact in stem cells marked by an arrow. Nucleus counterstained with 1 μg/ml 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI′). DP-MSCs, dental pulp mesenchymal stem cells.

DP-MSCs reached 70–80% confluency on the 8th–9th day of culture and BMSCs in 7–8 days. DP-MSCs showed a high proliferation rate (p < 0.001) compared with BMSCs from 2 to 7 days; but both reached a stationary phase later. Repeated measurement ANOVA showed overall significant difference between DP-MSC and BMSC over the entire time period. The mean PDT in 14 days for DP-MSCs and BMSCs was 31 ± 2.2 h and 51 ± 0.45 h, respectively (p < 0.001). For a cell density of 100 cells/60 mm plate, average number of colonies formed after 14 days of culturing was 50 ± 2 and 24 ± 1 for DP-MSCs and BMSCs (p < 0.001), respectively. Comparison of growth kinetics between two cell populations is shown in Fig. 3(a–c) and Table 1.

Fig. 3.

(a) Growth curve for DP-MSCs and BMSCs, (b) population doubling time (hr), (c) the number of colonies formed for a cell density of 100 cells, and (d) alkaline phosphatase activity of both the cell populations at days 7, 14, and 21. DP-MSCs, dental pulp mesenchymal stem cells.

Table 1.

Comparison of growth kinetics between DP-MSCs and BMSCs.

Cell growth assay
Observation	DP-MSC		BMSC		Repeated measure ANOVA—general linear model (GLM)
	Mean	±SD	Mean	±SD	Source	F
Day 1	10,666.67	500.00	10,000.00	0.00	Corrected model	838.48**
Day 2	14,500.00	750.00	11,333.33	1000.00	Intercept	39,800**
Day 3	24,000.00	866.03	17,333.33	4000.00	Group	640.12**
Day 4	32,000.00	866.03	23,333.33	2645.75	Days	1674**
Day 5	44,333.33	1000.00	33,333.33	4000.00	Group × days	27.34**
Day 6	68,166.67	1750.00	55,000.00	1887.46
Day 7	80,000.00	3968.63	60,000.00	3774.92
Day 8	82,166.67	3682.73	63,333.33	3307.19
Day 9	82,500.00	4520.79	66,000.00	3464.10

Population doubling time and clonogenic assay
Parameter	DP-MSCs		BMSCs		t value
	Mean	±SD	Mean	±SD
Doubling time (h)	31.11	2.22	51.83	0.45	18.680**
Clonogenic assay (no of colonies)	50.00	2.01	24.00	1.00	26.00**

**P < 0.001.

ANOVA, analysis of variance; DP-MSCs, dental pulp mesenchymal stem cells; SD, standard deviation.

Flow cytometry data demonstrated that more than 85% dental pulp stem cells expressed MSC-specific surface markers; CD44, CD73, CD90, CD105, and CD166, while the hematopoietic markers—CD11b, CD33, CD34 and CD45—were found only among <2% of the cells. Positive reaction was displayed by 7.2% of cells for Stro-1, an early MSC marker. Similar pattern of surface protein expression was seen with BMSCs also. Representative images of immunophenotyping are shown in Fig. 4 (a and b).

Fig. 4.

Flow cytometry analysis for the surface markers expression by (a) DP-MSCs and (b) BMSCs. Red histogram represents isotype control, while green histogram shows surface marker expression. In vitro differentiation assay of DP-MSCs: (c) osteogenic differentiation by alizarin red staining (arrow showing calcium deposits), (d) adipogenic differentiation by lipid droplets stained by Oil red O (arrow showing lipid droplets), (e) alcian blue staining showing chondrogenic differentiation, and (f–h) corresponding differentiation in the BMSCs. DP-MSCs, dental pulp mesenchymal stem cells.

Dental pulp and bone marrow MSCs showed differentiation to osteogenic, adipogenic, and chondrogenic lineages as shown in Fig. 4 (c–h). Cultures when stained with Oil red O showed lipid vacuoles (adipogenic induction) after 3 weeks. Osteogenic differentiation was observed by alizarin red–stained, calcium mineral deposits. ALP activity of cultures was measured over 21 days, in which peak expression was noted on day 14, which later decreased (Fig. 3d). BMSCs had significantly higher ALP activity in comparison to DP-MSC on day 7 and 14 (Table 2). Ability of both the cells for chondrogenic lineage was confirmed by alcian blue staining of sulfated mucosubstances. Negative controls did not show any differentiation.

Table 2.

Comparison of alkaline phosphatase activity between DP-MSCs and BMSCs.

Mean alkaline phosphatase activity (nmole/min/mg of protein)
Observation	DP-MSC		BMSC		Repeated measure ANOVA—general linear model (GLM)
	Mean	±SD	Mean	±SD	Source	F
Day 7	1302.00	22.96	1542.00	98.15	Corrected model	514.66**
Day 14	2224.00	21.00	2460.00	84.99	Intercept	24,890**
Day 21	1102.00	96.02	1164.00	87.43	Group	75.10**
					Weeks	1241**
					Group × weeks	8.04**

**P < 0.001.

ANOVA, analysis of variance; DP-MSCs, dental pulp mesenchymal stem cells; SD, standard deviation.

Discussion

Adult stem cells are widely researched for its therapeutic role in various tissue repair and regeneration applications. Although MSCs from bone marrow were initially studied for this purpose, the invasive collection procedures and less availability have precluded its widespread clinical use. A prelude for any novel alternate MSCs source would involve their characterization as well as evaluation of differentiation abilities. The International Society for Cellular Therapy (ISCT) 2006 guidelines were followed for MSC isolation from orthodontically extracted premolar teeth.¹⁹ The adherent monolayer formation of DP-MSCs in culture medium and the typical spindle morphology were akin to the BMSCs. The number of DP-MSCs in culture demonstrated a significant increase compared with BMSCs from day 2–7 (p < 0.001). On the 7th day, the cell numbers reached an eight-fold increase as to initial seeding. This was in accordance to a previous report that cited a six times rise in the proliferation rate of DP-MSCs isolated from deciduous teeth.¹⁸ Such steady rise in cell populations has been reported in the human dental pulp stem cells isolated from the third molar too.¹⁰

The favorable growth rate of DP-MSCs compared with BMSCs was further confirmed by the lesser PDT of the MSCs; 31.11 ± 2.2 h vs 51.83 ± 0.45 h (p < 0.001). This was within the range of growth rate previously reported for DP-MSCs isolated from the third molar teeth (20.79 h) and deciduous teeth (25.55 h).^{20, 21} These discrepancies in time periods may be due to the source from which MSCs were isolated and based on the number of cells seeded. The dependence on initial cell population on the proliferation rate has been understood by estimating PDT of deciduous teeth over 24 days at different densities.¹⁸ Cells plated at 1 × 10⁵/well displayed higher PDT, i.e., 80 h while lower cell density (1 × 10⁴/well) has shown a PDT of 50 h. Although the cell PDT has been found varying in different studies, the high expansion rate of MSCs from dental pulp-periodontal complex has so far been noteworthy. Our results from premolar teeth were in favor of it.

Other than PDT, the clonogenic efficiency was also significantly higher for the DP-MSCs (p < 0.001). DP-MSCs showed 50 colonies against BMSCs with 24 colonies. This was in harmony with the previous reports: 22–70 colonies for DP-MSCs from third molars whereas 2.4–3.1 colonies for BMSCs, when 10⁴ cells were initially plated.¹¹ More than 50% of cells were found capable of colony formation in studies by Eslaminejad et al²¹ (59% and 54.8% for DP-MSCs from the third molar and deciduous teeth, respectively) and Huang et al²² (72% and 83% DP-MSCs from mesiodens and deciduous teeth, respectively). Similar to proliferation rate, the variations in colony-forming capacity may also be attributed to the seeding cell number. As of now, there is consensus on the fact that clonogenic efficiency will be more when less number of cells is seeded.¹⁸ We noticed a consistent growth rate of DP-MSCs even after several passages. All these, a high cell growth rate with impressive colony-forming capabilities and lesser PDT of DP-MSCs, versus BMSCs, showed promising potential in regeneration therapies.

Further to the characterization, the cell immunophenotype was verified with antigenic markers, which are specifically expressed in the mesenchymal and hematopoietic stem cells. More than 85% of DP-MSCs expressed the positive mesenchymal surface proteins, CD44, CD73, CD90, CD105, and CD166. This was over and above the minimum criteria (CD73/90/105) proposed by the ISCT for MSCs. CD44 is a hyaluronan receptor that interacts with the components of extracellular matrix. Similarly, CD90 (Thy-1) and CD73 (ecto-5′-nucleotidase) are glycol-phosphatidylinositol–anchored proteins involved in the signal transduction and mediation of cell–cell and cell–matrix interactions. CD105 (endoglin) is a transmembrane receptor that binds to Transforming Growth Factor beta (TGF-β), and CD166 (Activated Leukocyte Cell Adhesion Molecule [ALCAM]) is an adhesion molecule.²³ The early mesenchymal marker, Stro-1, a transmembrane protein known for osteogenesis, showed positive reaction for 7.2% of cells. Surface marker results were in concurrence with the MSCs isolated from deciduous, third molar, apical papilla, and natal teeth.^{24, 25, 26, 27} In addition, DP-MSCs and BMSCs demonstrated negative reaction for hematopoietic markers: CD11b, CD33, CD34, and CD45. CD45 is a leukocyte marker, and CD34 is expressed in hematopoietic progenitors. CD33 is a key marker of myeloid cells/macrophages, and CD11b is a key marker of monocytes.¹⁸ Immunophenotyping reaffirmed the identity of MSCs.

Stemness is the most important characteristic of MSCs. In the present study, we were able to prove the competency of premolar pulp–derived MSCs to differentiate into osteogenic, chondrogenic, and adipogenic lineages. This was in accordance with some of the earlier studies on dental pulp stem cells.^{18, 20, 25} We also observed that the differentiation capacities of DP-MSCs were in par with that of BMSCs. Osteoblast differentiation of DP-MSCs and BMSCs was further evaluated by ALP activity to see their efficacy in bone regeneration. Although the ALP activity for BMSCs was higher than DP-MSCs (p < 0.001) at day 7 and 14, the pattern of rise and decline was alike for both. The ALP expression by premolar DP-MSCs was in agreement with a prior experiment performed using natal tooth stem cells. It mentioned a fourfold increase of ALP activity during 14 days of culture in osteogenic differentiation medium.²⁵ Related result has been found with deciduous teeth stem cells also within a period of 7 days.¹⁸ ALP activity is an early marker of osteogenesis, wherein the transcription and protein expression of the enzyme usually occurs from days 5–14 and thereafter the activity reduces, coinciding to the period of matrix mineralization.²⁸

To summarize, MSCs from premolar tooth (DP-MSC) extracted for orthodontic purposes demonstrated comparable growth characteristics with respect to BMSCs for cell growth rate, PDT, and clonogenic assays. They showed multilineage differentiation as well. More so, they concurred to all the known features of MSCs isolated from pulp periodontal complex. Considering the osteogenic differentiation and ALP expression of DP-MSCs, they are likely to find applications in scaffold-assisted bone tissue engineering procedures. Because these MSCs trace their origin to the neural crest cells; they are now also being investigated for neural and retinal tissue regeneration in the craniofacial area.²⁹ Therefore, developing a protocol for MSC isolation from orthodontically extracted premolars of healthy adolescents would be a major milestone in the efforts toward MSC banking and personalized stem cell–based therapies.

Conflicts of interest

The authors have none to declare.