Sample harvesting and transportation

Thirty six healthy premolars were obtained; they were donated by patients of the Naucalpan Orthodontic Specialty Clinic, who had previously signed informed consent forms according to the Bioethics Committee of the Iztacala Graduate School, attached to the National Autonomous University of Mexico (UNAM). The following inclusion criteria were used for tooth selection: only patients without systemic alterations, suitable periodontal health, first and second premolar teeth from right and left upper and lower jaws. Exclusion criteria were: patients afflicted with systemic diseases, patients with gingivitis and/or periodontal disease, premolars with presence of dental plaque and/or presence of caries and/or periapical reaction. Samples were transferred in 3 mm of D-MEM medium (BioWest) supplemented with 1% antibiotic (penicillin-streptomycin Gibco^® No Cl. 15140-122) at 4 ^oC.

Employed techniques are meticulously described in table I. In all seeding techniques complete D-MEM (BioWest) was used (added with L-glutamine, 10% of FBS and 1% of antibiotic, in standard culture conditions at 37 ^oC with presence of 5% CO₂.

Subcultures were performed with 60/80% confluence, after having been washed for 30 seconds with Hank solution (Sigma Aldrich); 500 μL of tripsin/ EDTA at 0.25%/0.1% (BioWest) during 1-3 minutes at a 37 ^oC temperature. Cell subcultures were conducted in a 1/3 proportion.

Cell proliferation assay

Five hundred cells were seeded in 24-well boxes, one cell to a well. Assessment of cell proliferation was conducted at 1, 3, 5, 7, and 14 days after seeding, this was accomplished with addition of 50 μL of MTT (Sigma Aldrich) incubated for four hours at 37 oC in a 5% CO₂ atmosphere. At a later point, 100 μL of dimethyl sulfoxide (DMSO, Sigma Aldrich) were added in order to achieve optic density reading in a spectrometer at al 540 nm wave length.

Cell migration assessment

With cells in confluence, a lesion was performed along the culture, causing continuity loss, the culture was monitored with photographs taken at 24, 48 and 72 hours.

Immunocytofluorescence

Cells were fixated in a 4% paraformaldehyde phosphate tampon for 20 minutes. Blocking was conducted in PBS with 3% bovine serum albumin and 0.01% Triton-X 100. Cells were stained with the following: anti-human rabbit antibodies conjugated with Fluor^® 647: PE, TRIT, FITC, and DAP (Fluoroshield^®), RUNX2, osteocalcin, CD73, CD90, CD105 and vimentin (1:250 Santa Cruz) during 45 minutes. Samples were examined in a Zeiss Axio LAB1 fluorescence microscope.

Osteogenic differentiation

In this process, 105 were seeded in passage 3; medium StemPro Osteogenesis Differentiation Kit (Gibco^®) was added; it was replaced every 72 hours for 14 days.

Staining with alizarin red

Once the cells were differentiated after 7 and 14 days’ culture, they werefixated and stained with alizarin red. They were then subjected to three five minute rinses with PBS; 500 μL 0.1% alizarin red were added (HYCEL®) and were left to rest for 15 minutes at room temperature.

Cell population harvesting

Poor technical specificity of found articles influenced in obtaining cell population harvesting, cultures conducted in triplicate with each of the studied techniques lead us to obtain a minimum cell concentration which did not achieve confluence. In some cases, after a culture time of seven days, no cells were obtained; and in other cases, the scarce amount of obtained cells was susceptible to contamination, probably caused by the transport medium of the tooth and the washes.

Optimum cell population for the study was achieved using the «standardized technique of enzymatic digestion and explant culture». Once the periodontal tissue was dissected and seeded, cells appeared in the first 72 hours, which after an additional period of 46 hours showed fibroblastoid aspect in the cultured explants periphery, filling thus the culture dish in 65 to 70%. After this, explants were eliminated with the washes. After seven days, the culture dish was found to exhibit cells in confluence (Figure 1).

Figure 1 Image A) after 72 hours culture, cell growth can be observed around the explant. Image B) taken after 8 days of confluence culture. 

Viability and/or cell proliferation

Cell population self-renovation capacity was assessed by means of methylthiazole tetrazolium assay (MTT) 3-(4,5-dimethylthiazole 2-il)-2,5diphenyltetrazolium bromide; spectometry-measured absorbance was proportionate to cell proliferation viability.

Five hundred cells were seeded in 24-well plates; they were evaluated at 1, 3, 5, 7 and 14 days. Absorbance was measured at a 540 nm wave length. Obtained measurements were incorporated in a graph. An ascending graph was obtained, which corresponded to a concentration of cells per measuring unit, thus demonstrating that cells preserved their proliferation during 14 days in standard culture circumstances (Figure 2).

Figure 2 Average results obtained from reading at 540 nm wavelength. 

Cell migration capacity

Cell migration towards the injured area was observed after 12 hours of lesion onset; cells coated between 10-20% of the critical area. After 24 hours, cell numbers increased, covering then from 40-60% of the surface of the culture jar. After 48 hours, cells were observed to be in semi-confluence; after 72 hours, cells totally covered the area, this can be observed in images of figure 3.

Figure 3 Photograph taken immediately after establishing lesion in the culture. B) 12 hours after the lesion, migration of a group of cells re-colonizing 10 to 20% of the lesion can be observed. C) 24 hours later migration of a cell group was observed which re-colonized damaged area in 40/60%. D) and E) After 48 hours, it can be observed that cells have covered 80-90%. F) 72 hours after the lesion, cells reach confluence once again. 

Immunofluorescence

Immunofluorescence assays were performed in the third subculture of our cell population. After counting, positive expression to CD90 was observed in over 80% of all cells, positive expression to CD73 was observed in 70%, positive expression to CD105 in 60% and vimentin in 90% of the population (Figure 4).

Color image available at: www.medigraphic.com/facultadodontologiaunam

Figure 4 Images taken with fluorescence microscope at 40x amplification. A) CD90 expression (green), B) Nuclear DAPI (blue), and C) Fusion, D) CD73 (red), E) Nuclear DAPI (blue) and F) Fusion, G) Vimentin (green), H) CD105 (red) and I) Fusion of both with nuclear DAPI (blue). 

Osteogenic differentiation

Once bone differentiation was induced, cells lost their fibroblastoid morphology in seven days, and acquired a less elongated, cuboid-like prismatic morphology. There was also a greater amount of extracellular matrix as well as presence of calcification nodules, which were observed by means of staining with alizarin red the cultured area, revealing thus a size of 9.6 cm² (Figure 5).

Color image available at: www.medigraphic.com/facultadodontologiaunam

Figure 5 Staining with alizarin red A) macroscopic photograph with contrast with hematoxylin of control cells and cells induced to differentiation after 14 days. Staining with alizarin red of differentiated cells B) 10x, C) 40x. 

RUNX2 is a transcription factor indispensable for bone maturation, it was expressed in our cell population in osteogenic differentiation at 14 days, in at least 15% of all cells.

Osteocalcin is a protein secreted at late stages of bone differentiation, it is able to bond to calcium for its stabilization. The presence of this molecule in 90% of cells after 14 days indicates mineralization regulation (Figure 6).

Color image available at: www.medigraphic.com/facultadodontologiaunam

Figure 6 Images taken with fluorescence microscope. A) Expression of RUNX2, 40x (green), B) Nuclear DAPI (blue) 40x and C) Fusion 40x, D) Osteocalcin (green), E) Nuclear DAPI (blue) 63x and F) Fusion 63x.