Purification of Stem Cells from Oral Pyogenic Granuloma Tissue

2.1. Preparation of Single-Cell Suspension from Pyogenic Granuloma

Four samples of pyogenic granuloma were collected from the patients referring to Shiraz School of Dentistry. All samples were located on gingiva, and an experienced pathologist confirmed the diagnosis through pathological sections (Fig. 1). The patients signed an informed consent before being enrolled in the study. The study was in accordance with the Declaration of Helsinki and was approved by the local ethics committee.

The samples of pyogenic granuloma tissue were transferred to PBS-EDTA solution with 1% penicillin/streptomycin and 1% fungizone (both from Gibco/Invitrogen Company, Carlsbad, California, USA). The tissues were broken under sterile conditions by using enzymatic digestion while occasionally shaking for 1 hour in a solution of 3 mg/mL collagenase type 1, and 4 mg/mL dispase type 2 (both from Sigma, St. Louis, Missouri, USA). The resulting single-cell suspension was passed through a 70-micron cell filter (Biosciences BD, San Jose, California, USA) and centrifuged for 10 minutes to remove the supernatant remaining enzymes. The cells were re-suspended in the environment and each sample was separately used for the next steps including evaluation of the potential of colonization or cultivation for further analysis.

2.2. Isolated Cells Culture

The single-cell suspensions were seeded onto cultivation flasks within the α-MEM along with 4 mM GlutaMax, 100 U/ml penicillin, 100 mcg/ml streptomycin and 20% FBS (all from Gibco/Invitrogen). The cells were cultured for 72 hours at 37°C in 5% CO₂ and 90% humidity. Then, the free cells and residuals were removed and fresh medium was added to the adherent cells. The medium was changed twice a week until the flask reached 80% confluence. The cells were then released with Trypsin- EDTA (Gibco/Invitrogen) and the second culture was performed. They were passaged three times before they could be used for differentiation analysis or flow cytometry.

2.3. Colony Forming Unit Assay

To evaluate the efficiency of the derived single-cell colonization (colony-forming unit fibroblast assay [CFU-F]), the primary cells derived from the pyogenic granuloma were cultured on six-chamber plates with the living cell concentration of 1000 cells per ml from the culture medium. The single-cell derived colonies were defined as those units with more than 50 cells.

2.4. Adipogenic Differentiation

As formerly mentioned, the third passage cells were collected for adipogenic differentiation and cultured in MesenCult medium along with 10% adipogenic stimulatory supplements (both from STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) with a density of 2×10³ cells in a milliliter of medium within the two-chamber cultivation slides according to the manufacturer's instructions. The cells were cultured for 3-4 weeks. Half of the media were changed only when the ambient color turned yellow. When the cells exhibited appropriate morphological changes, they were analyzed by using Oil Red staining (Sigma). In order to perform the staining, the cells were incubated in 4% formalin containing 1% calcium chloride for one hour. The cells were then stained with Oil Red O solution for 10-15 minutes, and in ethanol 70% for one minute; and then, washed with distilled water.

2.5. Osteogenic Differentiation

For osteogenic differentiation, the third passage cells were collected and cultured in NH OsteoDiff medium (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) with a density of 10⁴×3 cells in a milliliter of the medium according to the manufacturer's instructions. The medium was replaced twice a week for three weeks. To verify the differentiation, after suitable morphological changes, the cells were washed with PBS and incubated in methanol for 10 minutes. They were stained with 1.0 M solution of red Alizarin in 25% ammonia water for 24 hours and washed with distilled water.

2.6. Flow Cytometry

Analysis of the cell surface antigen scenarios was done by using the cells obtained from the third passage. The isolated cells were incubated for 10-30 minutes in a dark environment with the following anti-human antibodies: CD90- fluorescein isothiocianate (FITC), CD 34- phycoeritrin (PE), CD 45- Peridinin chlorophyll protein (PerCP), CD 271-FITC (MiltenyiBiotec), CD106-PE, CD 44-FITC, CD73-PE, CD14-FITC (BD Biosciences), and CD105-PerCP (AbDSerotec, Kidlington, Oxford, England). Isotype-matched irrelevant monoclonal antibodies such as Mouse IgG1-PE, IgG2a-FITC (AbDSerotec, Kidlington, Oxford, England), IgG2a-PerCP, IgG2b-FITC and IgG2a-PE (Miltenyi Biotec) were used to omit the effect of non-specific cells staining. Flow cytometry analysis was done in a FACS Calibur device (BD Biosciences) by using the cell quest as data acquisition software. Win MDI software, version 8.2, was employed for data analysis.

3.1. Culture Specifications

The pyogenic granuloma-derived cells attached to plates two to five days after the primary culture. These cells reached confluence in 12-21 days, with a proper appearance similar to spindle-shaped fibroblast (Fig. 2A). They could also successfully form single-cell derived colonies (Fig. 2B).

3.2. Colony Forming Unit Assay

To assess single cell-derived colony formation (Colony Forming Unit-Fibroblasts [CFU-F assay]), only colonies with more than 50 cells were considered in colony enumeration (Fig. 2B). From 1000 plated pyogenic granuloma tissue-derived cells 11.3 ± 8.2 colonies were formed.

3.3. Differentiation Analysis

Adipogenic differentiation was confirmed through morphological changes and the corresponding staining. One week after cultivation in adipogenic medium, the cells showed small isolated vacuoles whose number and size increased over time, and all of them were stained with Oil Red O (Fig. 2C). The earliest evidence of differentiation into osteoblasts matrix sediments was observed during the second week. Full differentiation into osteoblast cells lasted four weeks. Alizarin red staining documented the mineralization (Fig. 2D).

3.4. Flow Cytometry Results

Analysis of the flow cytometry of the cells obtained from cultures of pyogenic granuloma-derived cells showed that the cells were positive for mesenchymal markers such as CD 44, CD 271, CD 90, CD 73, and negative for the surface molecules like CD 14, CD 34 and CD 45. These show a heterogeneous phenotype for CD105 and CD106 (data from one of the samples are shown in Fig. (3)).