Minimal influence of chronic inflammation on the potency and differentiation characteristics of gingiva-derived mesenchymal stem cells-An in vitro study.

OBJECTIVE: Gingiva-derived mesenchymal stem cells (GMSCs) have been identified and characterized from healthy tissues. However, reports on the influence of chronic inflammation on their stemness characteristics are sparse. The present study evaluated the potency and differentiation ability of GMSCs from periodontally healthy GMSC (H-GMSC) and inflamed GMSC (I-GMSC) tissues.
MATERIALS AND METHODS: Established H-GMSCs and I-GMSCs were evaluated on their potency characteristics, such as morphology, viability, proliferation rate, population doubling time, colony-forming ability, expression of stemness markers, and mesenchymal differentiation potential.
RESULTS: H-GMSCs and I-GMSCs exhibited fibroblast-like morphology and showed >95% viability with high proliferation potential and shorter doubling time. H-GMSCs showed fewer and smaller colonies, whereas I-GMSCs exhibited multiple and larger colonies. The evaluation of stemness markers revealed that both H-GMSCs and I-GMSCs were weakly positive for stage-specific embryonic antigen-4, Stro1, and CD105 (Endoglin), strongly positive for CD73 and CD90, and negative for the hematopoietic cell markers, CD34 and CD45. H-GMSCs showed a slightly higher osteogenic potential when compared to I-GMSCs, while I-GMSCs had a higher adipogenic potential than H-GMSCs.
CONCLUSION: The findings showed that the inflammatory environment might have a stimulatory effect on the growth kinetics and ability of colony formation in GMSCs. However, varied osteogenic and adipogenic differentiation was observed between H-GMSCs and I-GMSCs. Copyright:

INTRODUCTION

Periodontitis is a chronic inflammatory disease characterized by progressive destruction of tooth-supporting structures. The ultimate goal of periodontal therapy is the preservation of the dentition in function and esthetics. This is achieved by resolving inflammation and regenerating lost tooth-supporting structures. Current clinical approaches toward regeneration of periodontal tissue have been met with varying success, because of the inability to control an aberrant inflammation and scanty progenitor cells present at the defect site.[1] Thus, alternative strategies using biologic mediators could overcome these drawbacks.[2]

Among the several approaches, cell-based therapy to promote regeneration in a predictable manner has gained attention. Mesenchymal stem cells (MSCs) with their ability of self-renewal, multilineage differentiation, and immunomodulatory properties are regarded as valuable sources for tissue regeneration.[3] Evidence suggests that MSCs exist in dental tissues.[4] The isolation and characterization of human gingiva-derived mesenchymal stem cells (GMSCs) were first reported in 2009,[5] and these cells exhibited clonogenicity, self-renewal, and multilineage differentiation capacity.[567] GMSCs are derived mainly from cranial neural crust cells and have shown characteristics of pluripotent stem cells.[89] The gingival connective tissue has been considered an alternative source of MSCs for periodontal regeneration due to their immunomodulatory properties in vitro and in vivo,[591011] rapid ex vivo expansion, ease of isolation, and substantial availability.[81213]

In patients undergoing treatment for periodontal disease, the source of GMSCs is most likely the inflamed gingival tissue. Previous studies have identified MSC-like cells in diseased dental tissue.[141516] MSCs isolated from inflamed pulp and periodontal ligament retained their regenerative potential but exhibited dysfunctional immunomodulatory properties. Studies evaluating the influence of inflammation on GMSCs demonstrated fewer inflammation-related changes.[7141718] However, it was showed that inflammation reduced their population doubling time (PDT).[14] Besides, inflammatory environment induced the GMSCs to differentiate toward a profibrotic phenotype, while still retaining a high proliferative activity.[1519]

Studies so far have reported contradictory results with the influence of inflammation on the biological properties of GMSCs. To better understand the impact of local inflammatory microenvironment, the present study evaluated the potency and differentiation ability of GMSCs isolated from periodontally healthy GMSC (H-GMSC) and inflamed GMSC (I-GMSC) tissues.

MATERIALS AND METHODS

This study followed the Declaration of Helsinki on medical protocol and ethics, and was approved by the Institutional Ethics Committee and the Institutional Committee for Stem Cell Research. Healthy gingival tissues were collected from individuals when they underwent routine extraction for orthodontic reasons, crown lengthening procedure, or during planned third molar removal. Gingiva from inflamed tissue was obtained following an access flap surgery from patients clinically diagnosed with chronic periodontitis according to the American Academy of Periodontology Classification of disease.[14] The informed consent was obtained from the patients prior to the tissue collection, and the tissues that were normally discarded following the surgical procedure were used for the study. A total of five tissue samples from each group were harvested for establishing the cell lines with minimal heterogeneity.

GMSCs were isolated from periodontally healthy and inflamed tissues by partial enzyme digestion method following previously published protocol with minor modifications.[14] Briefly, collected tissues were washed thrice with Dulbecco's phosphate-buffered saline (Gibco, Life Technologies, Grand Island, NY, USA) and minced into 1–2 mm pieces. The tissue remnants were then incubated in 0.1% collagenase type IV enzyme (Gibco) for 1 h, and enzyme-digested samples were centrifuged at 1000 rpm for 5 min. Then, 4–5 tissue explants were placed overnight with minimum amount of growth medium (Dulbecco's Modified Eagle's Medium [DMEM]-high glucose, Gibco) consisting 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C in a humidified atmosphere of 5% CO2 in air. The culture was continued till the cells reached 70%–80% confluence, and they were detached using 0.1% (w/v) trypsin-ethylenediaminetetraacetic acid (Gibco) solution. Cells were then subpassaged 3–5 times with each passage duration of 12–15 days for further analysis. During the culture, cells were observed for attachment and morphology under a phase-contrast microscope (Olympus, Tokyo, Japan).

The percentage of live cells was calculated at every passage of GMSCs from Passage 1 (P1) to P5. Cell viability was assessed by 0.4% trypan blue (Gibco) staining using hemocytometer. Cells which stained blue were considered as dead cells and transparent cells were counted as live cells.

Proliferation and PDT of GMSCs were assessed by plated 5000 cells/well in a 12-well tissue culture plate (Thermo Scientific, USA) in triplicates. Every 3 days for 12 days, cells from each well were detached and counted with a hemocytometer. The culture medium was changed every 3 days. PDT of GMSCs was calculated using a formula: PDT = t (log2)/(log Nt−log No) where t represents the culture time and No and Nt are the cell numbers before and after seeding, respectively.

For assessing the potency and differentiation ability of GMSCs, the previously described methods were followed with minor modifications.[8] Colony-forming ability assay was done by culturing GMSCs in a 12-well plate at 50 cells/cm2 for 15 days. The media was changed once in 3 days. For staining, cells were prefixed with 3.7% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), stained with 1% Giemsa stain (Sigma-Aldrich) for 20 min at room temperature, and observed under a microscope (Olympus).

Flow cytometry analysis was performed by fixing the GMSCs in 3.7% paraformaldehyde (Sigma-Aldrich) for 30 min after reaching ~80% confluence. Primary antibodies, such as Alexa Fluor-488-conjugated anti-mouse CD105 (Biolegend, CA, USA), were labeled directly at 37°C for 1 h, and unconjugated stage-specific embryonic antigen-4 (SSEA4, eBioscience, CA, USA), Stro1 (eBioscience), CD73 (Biolegend), CD90 (eBioscience), CD105 (Endoglin, Biolegend), CD34 (Biolegend), and CD45 (eBioscience) were incubated for 2 h at 37°C. Fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (eBioscience) was used as a secondary antibody and incubated for 1 h at room temperature. The standard was established by isotype-matched control (eBioscience). A total of 10,000 cells were acquired and analyzed by a BD FACSCalibur (Becton Dickinson, NJ, USA) with CellQuest software. (Becton Dickinson, NJ, USA).

For osteogenic differentiation, H-GMSCs and I-GMSCs were cultured in DMEM supplemented with 0.1 μM dexamethasone (Sigma-Aldrich), 10% FBS, 10 mM β-glycerol phosphate (Sigma-Aldrich), and 0.2 mM ascorbic acid (Sigma-Aldrich) for 3 weeks. The medium was changed twice a week. Osteogenesis was assessed after the completion of induction with von Kossa (Sigma-Aldrich) staining method.

For adipogenic differentiation, cells at 70% confluency were cultured in DMEM supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), 0.1 mM indomethacin (Sigma-Aldrich), 10 μg/mL insulin (Sigma-Aldrich), and 10% FBS (Gibco) for 3 weeks. Medium changes were carried out twice weekly, and adipogenesis was assessed after 3rd week of induction. The presence of lipid droplets was evaluated by staining with 0.5% w/v Oil Red O (Sigma-Aldrich).

Statistical analysis

All data were expressed as the mean ± standard deviation from at least three independent experiments. Analysis of variance was performed by GraphPad Prism software (GraphPad, CA, USA) with Tukey's post hoc test. P < 0.05 was considered to be statistically significant.

RESULTS

The initiation of release of H-GMSCs and I-GMSCs from the tissue explants was observed from 24 h to 48 h. The cells emerged out clearly during the 5th day of tissue placement on the culture dish [Figure 1a and d]. Initially, both GMSCs exhibited a heterogeneous mixture of cells with slightly varied morphology. Later, plastic adherent GMSCs showed a characteristic fibroblast-like morphology by day 10 of culture [Figure 1b and e]. On day 15, both GMSCs reached 80%–90% confluency with cells displaying long, slender morphology [Figure 1c and f].

Figure 1

Primary culture and morphological characteristics of H GMSCs (a-c) and I GMSCs (d-f); (a and d) Cells emerging out from tissue explant on the 5th day of its placement on the culture dish (arrows); (b and e) Plastic adherent cells exhibited proliferative activity after attaining characteristic fibroblast like morphology at 10 days of culture; (c and f) On day 15, both GMSCs reached >80% confluency with cells showing long, slender morphology. Images: ×10. H GMSC – Gingiva derived mesenchymal stem cells obtained from healthy tissue; I GMSCs – Gingiva derived mesenchymal stem cells obtained from inflamed tissue

H-GMSCs and I-GMSCs showed >95% viability at each passage analyzed [Figure 2], and no significant differences (P > 0.05) in percentage values were observed between the cell lines at different passages.

Figure 2

Viability of H-GMSCs and I-GMSCs. GMSCs from both the groups showed >95% viability at each passage (P1 to P5) with no significant differences (P > 0.05). Values are represented as means ± standard deviation of triplicates at each passage. H-GMSC – Gingiva-derived mesenchymal stem cells obtained from healthy tissue; I-GMSCs – Gingiva-derived mesenchymal stem cells obtained from inflamed tissue

Cell proliferation assay was performed by counting the cells at days 0, 3, 6, 9, and 12 using a hemocytometer, and the results are depicted in Figure 3a. There was a significant (P = 0.0187) difference in the proliferation rate between H-GMSCs and I-GMSCs on day 9 of culture. Further, cell density on day 12 was higher in I-GMSCs than in H-GMSCs. However, no significant (P > 0.05) difference in PDT values was observed between H-GMSCs and I-GMSCs [Figure 3b]. The average PDT values of 65.5 h and 52.3 h were recorded for H-GMSCs and I-GMSCs, respectively.

Figure 3

Proliferation and PDT analysis of H-GMSCs and I-GMSCs. (a) Both cells were highly proliferative with no significant difference (P > 0.05) between day 0 and day 3. However, significant difference was observed on day 9 (*P < 0.05). Values are represented as means ± standard deviation of triplicates at each time interval; (b) No significant difference (P > 0.05) in PDT was observed between H-GMSCs and I-GMSCs. H-GMSC – Gingiva-derived mesenchymal stem cells obtained from healthy tissue; I-GMSCs – Gingiva-derived mesenchymal stem cells obtained from inflamed tissue; PDT – Population doubling time

The colony-forming ability of GMSCs reflected the prevalence of stromal clonogenic precursors in gingival tissue. H-GMSCs exhibited fewer and smaller colonies [Figure 4a], whereas I-GMSCs showed multiple and larger colonies [Figure 4b]. Aggregates of 50 or more cells were scored as colony-forming unit-fibroblast colonies.

Figure 4

Colony-forming assay of H-GMSCs and I-GMSCs. Giemsa indicating the colony-forming ability of H-GMSCs (a) and I-GMSCs (b) (arrows). H-GMSCs showed fewer colonies, whereas I-GMSCs exhibited a more number of colonies. Images: ×10. H-GMSC – Gingiva-derived mesenchymal stem cells obtained from healthy tissue; I-GMSCs – Gingiva-derived mesenchymal stem cells obtained from inflamed tissue

Results of flow cytometry analysis are presented for H-GMSCs [Figure 5a] and I-GMSCs [Figure 5b]. The expression of SSEA4 and Stro1 in both GMSCs was slightly weaker and varied in intensity. However, H-GMSCs and I-GMSCs displayed a high expression of markers associated with MSCs, such as CD73 and CD90, in contrast to hematopoietic cell markers, CD34 and CD45, which showed very low expression. A higher expression of CD73 and CD90 in I-GMSCs indicated their role in maintaining stemness properties. The expression of CD105, a marker associated with lineage-committed cells, was weak in both GMSCs.

Figure 5

Flow cytometry analysis of (a) H-GMSCs and (b) I-GMSCs. Cells were stained with respective antibodies as indicated. IC indicates isotype control. In merged images, dark-lined histograms indicate signal of isotype control and green-lined histograms show the reactivity with indicated marker. Percentage expression of each marker is presented. H-GMSC – Gingiva-derived mesenchymal stem cells obtained from healthy tissue; I-GMSCs – Gingiva-derived mesenchymal stem cells obtained from inflamed tissue. CD – Cluster of differentiation, SSEA – Stage-specific embryonic antigen, FITC – Fluorescein isothiocyanate, GH – Gingiva-healthy, GI – Gingiva-inflamed, PE – Phycoerythrin, Stro1 – Stromal cell surface marker 1

The results of osteogenic potential of H-GMSCs and I-GMSCs are presented in Figure 6a-d. Both GMSCs in control maintained a fibroblastic morphology throughout the culture period of 21 days [Figure 6a and c]. Upon osteogenic induction, cell size reduced and turned irregular by forming a slightly more polygonal shape. Later, the deposition of mineralized nodules in the cultures was confirmed by von Kossa staining in H-GMSCs [Figure 6b] and I-GMSCs [Figure 6d]. H-GMSCs showed a slightly higher osteogenic potential when compared to I-GMSCs.

Figure 6

Osteogenic differentiation H-GMSCs and I-GMSCs. Cells with osteogenic induction showed deposition of calcium mineralized matrix (arrows) as evidenced by von Kossa staining (b, H-GMSCs and d, I-GMSCs); GMSCs in control exhibited fibroblast-like morphology (a, H-GMSCs and c, I-GMSCs). Images: ×10 and × 20. H-GMSC – Gingiva-derived mesenchymal stem cells obtained from healthy tissue; I-GMSCs – Gingiva-derived mesenchymal stem cells obtained from inflamed tissue

The results of adipogenicity in H-GMSCs and I-GMSCs are presented in Figure 7a-d. Small vacuoles appeared in GMSCs of monolayer cultures treated with adipogenic induction medium [Figure 7b and d] compared to their absence in untreated control [Figure 7a and c]. On day 21, Oil Red O staining confirmed the presence of neutral lipid vacuoles, consistent with an adipocyte phenotype. I-GMSCs showed a marginally greater adipogenic potential when compared to H-GMSCs.

Figure 7

Adipogenic differentiation H-GMSCs and I-GMSCs. Cells cultured under adipogenic conditions indicated the presence of neutral lipid-positive vacuoles (arrows), consistent with adipocyte phenotype (b, H-GMSCs and d, I-GMSCs). GMSCs in control remained as fibroblast-like cells (a, H-GMSCs and c, I-GMSCs). Images: ×10 and × 20. H-GMSC – Gingiva-derived mesenchymal stem cells obtained from healthy tissue; I-GMSCs – Gingiva-derived mesenchymal stem cells obtained from inflamed tissue

DISCUSSION

GMSCs might be an alternative source for cell-based therapy and tissue engineering due to their ease of access compared to the progenitor cells of the periodontal ligament, which have an innate ability to differentiate into various tissues of the tooth-supporting structures.[20] The source of GMSCs for cell-based therapy in patients diagnosed with generalised periodontitis is the inflammed gingiva. It has been speculated that constant exposure to oral microflora and mechanical stimulation during mastication, may have modulated these cells to acquire the ability to preserve their MSC properties and resist infection in the presence of an inflammatory microenvironment.[9] However, the influence of prolonged inflammation on the potency of GMSCs is inconclusive.

The MSC derived from gingiva has been hypothesized to possess properties distinct from other dental MSCs due to the unique microenvironment to which they are constantly exposed.[9] In the present study, GMSCs were successfully isolated from periodontally healthy and diseased/inflamed gingival tissue by partial digestion using 0.1% collagenase type IV enzyme that enabled early release of cells with plastic adherence ability. Previous studies employed dispase enzyme in combination with collagenase for obtaining cell suspension and established the primary cultures.[5812] In our study, semi-digested gingival tissue, which was used as an explant, yielded more number of H-GMSCs and I-GMSCs with greater homogeneity by day 15 of primary culture. It is suggested that a shorter incubation period with a single enzyme minimized the effect on GMSCs, yielding a higher number of cells.

In vitro self-renewal capacity of GMSCs is customarily established by cellular and growth kinetic parameters, including viability, proliferation, and doubling time. These results provide valuable data for the prospective application of GMSCs in therapy. In this study, GMSCs from both the groups showed >95% viability from P1 to P5. Further, these cells were highly proliferative and had a shorter PDT during culture expansion. The colony-forming assay is an indicative of the quality of MSCs present within the tissue, while the proliferation rate and doubling time show their ability for culture expansion. Although the I-GMSCs exhibited multiple and larger colonies than H-GMSCs, there was no significant difference in the number of adherent CFU-F. Enhanced proliferation rate may be the reason for the formation of a slightly higher number of colonies in I-GMSCs. Previous studies demonstrated that GMSCs compared to periodontal ligament stem cells have a more effective clone forming ability.[17] Hence, it was speculated that inflammation might have a stimulatory effect on I-GMSCs. These findings are in accordance with previous studies that reported a stimulatory influence of inflammation on the proliferative ability of GMSCs.[7141517]

MSCs comprise a heterogeneous population with different lineage commitments, which may be related to their in vivo environment.[9] The evaluation of MSC surface markers revealed that both H-GMSCs and I-GMSCs were weakly positive at varied intensity for SSEA4, Stro1, and CD105, strongly positive for CD73, and CD90, and negative for the hematopoietic cell markers, CD34 and CD45. The immunophenotypic profiles confirmed the stromal origin of GMSCs in our culture. H-GMSCs and I-GMSCs exhibited a strong positivity for CD73 and CD90. Higher expressions of these markers are in agreement with previous observations.[581221]

Stro1 has been suggested to be involved in clonogenicity and may facilitate homing and angiogenesis of MSCs. It is as yet unclear if Stro1 expression correlates with multipotency. SSEA4 is an embryonic stem cell marker, indicative of the clonogenicity and multipotency of MSCs.[22] Further, more Stro1- and SSEA4-positive MSCs were observed in periodontally healthy than in diseased gingival tissue.[15] However, our results showed a mild-to-low positivity for SSEA4 and Sto1 with no significant difference in their expression between the health and disease groups. The low positivity of Stro1 in our study may be due to the gradual loss of Stro1 expression with culture.[22] While MSCs are strongly positive for CD105, our study demonstrated a low positivity for this marker in healthy and diseased gingival tissues. Collectively, the results suggest that chronic inflammation did not affect the expression of stem cell markers as reported previously.[141517]

We observed that I-GMSCs had decreased osteogenic potential with higher adipogenic differentiation ability and retained their proliferative features. Similarly, in earlier studies, MSCs from inflamed periodontal ligament showed a decreased capacity for mineralized nodule formation when compared to those from healthy tissue.[23] It was suggested that, during periodontitis, secretion of excessive pro-inflammatory cytokines can modulate the osteoblastic differentiation of MSCs through different regulatory mechanisms, such as inhibition of nuclear factor-kappa B through β-catenin signaling which represses miR-21, and/or by downregulation of RUNX2.[2425] However, a recent study showed that the osteogenic ability of GMSCs from the inflamed tissue was preserved when cultured in cytokine-preconditioned media.[26]

CONCLUSION

The results of the present study validated that GMSCs can be isolated from both healthy and diseased/inflamed gingival tissues, and the inflammatory environment might have a stimulatory effect on the growth kinetics and ability of colony formation in GMSCs. Further, I-GMSCs showed reduced osteogenesis and enhanced adipogenic potential compared to H-GMSCs. However, further research is warranted to evaluate the potential of GMSCs obtained from periodontitis patients using animal models for cell-based therapies.

Financial support and sponsorship

This work was supported by Nitte (Deemed to be University) faculty research grant.

Conflicts of interest

There are no conflicts of interest.