The effect of platelet lysate in culture of PDLSCs: an in vitro comparative study

Sample collection

After study description, signed informed consents (Form- S1) were obtained from all donors before inclusion in this study. Third molar teeth were collected from three donors aged 18, 19 and 21 years. All donors showed no clinically evident disease or history of medications, smoking or alcohol consumption.

Platelet lysate preparation

Sample collection was performed according to the Institutional Review Board (IRB) guidelines from the Cell Therapy Center/University of Jordan (99/2014/TRBJ). Blood samples were collected in Citrate tubes (BD Vacutainer, USA) and centrifuged at 192 xg for 13 min at 4 °C (3-16KL; Sigma, St. Louis, MO, USA). After centrifugation, supernatants were pooled from multiple donor samples and frozen at −80 °C until further use. To prepare platelet rich plasma (PRP), pooled samples were centrifuged at 192 xg for 10 min and followed by two rapidfreeze-thaw cycles (−80 °C and 37 °C). The resulting human PLwas centrifuged for 10 min at 3,894 xg and 4 °C to remove the platelet bodies. And finally, the PL was filtered through 0.22 µm filters (TPP, USA) to remove the platelet membranes (Abuarqoub, Awidi & Abuharfeil, 2015). 

PDLSCs isolation

PDL tissues were either undergone enzymatic dissociation or explants culture for the isolation of PDLSCs.

1. Enzymatic dissociation method

For enzymatic digestion, first the teeth were disinfected with PBS containing 10% Pencillin / Streptomycin (Gibco, Waltham, MA, USA). Then, PDL tissue was dissected from the mid third level of the root and digested in a solution containing 3 mg/mlcollagenase type I (GIBCO, Waltham, MA, USA) and 4 mg/ml dispase (GIBCO, Waltham, MA, USA) for 1 h at 37 °C. Single-cell suspensions were obtained by passing the cells through 70 µm cell strainer (BD, USA) and then cells were centrifuged at 1,000 rpm for 5 min. The resulting pellet was re-suspended in 1 ml of cell culture media, seeded at 5,000 cells/cm² seeding density in 6-well plates and maintained in alpha-Modification ofEagle’s Medium(α-MEM, GIBCO, USA) supplemented with 100 µg/ml penicillin/streptomycin (Invitrogen, Waltham, MA, USA), 2 mM L-glutamine (Invitrogen, USA), 0.25 µg/ml Amphotericin B (Invitrogen, Waltham, MA, USA) and 5% PL. The cells were incubated at 37 °C in 5% CO₂, and medium was exchanged every three days until the cells reached the confluency. The morphological appearance was observed under the inverted microscope (Zeiss, Cincinatti, OH, USA), images were taken on day 7 of the primary culture. 

2. Explant method

Each PDL tissue sample was cut into 1–2-mm fragments, and individual pieces were placed in a 6-well culture plate with a minimal amount of α-MEM medium supplemented with PL. Plates were kept in the incubator at 37 °C and 5% CO₂ till the attachment of tissue fragments. Medium was exchanged every 3 days until the outgrowth of the PDLSCs was observed. The morphological appearance was observed under the inverted microscope (Zeiss, USA), images were taken on day 7 of the primary culture. 

Immunophenotyping using flow cytometry

To assess the expression of MSC surface markers, PDLSCs were harvested at passage 3 and washed twice with PBS. Approximately, 2 × 10⁶ cells were incubated for half an hour at room temperature (RT) with fluorescin-labeled antibodies using the mesenchymal stem cell characterization kit (stem flow kit; BD Biosciences, Franklin Lakes, NJ, USA). The kit contains antibodies for the following MSC markers: PerCP Cy5.5-CD105, FITC-CD90, PE-CD44 and APC-CD73 and the PE-Negative cocktail hematopoietic stem cell (HSC) markers (CD34, CD45, Cd11b and HLA-DR) and their isotype controls. After incubation, the cells were centrifuged at 300 xg for 5 min andthen re-suspended in PBS. Antibodyconcentration used was based on the manufacturer’s instructions (BD, Franklin Lakes, NJ, USA). The expression profile was analyzed by Flourescience Activated Cell Sorter FACS Canto II (BD, Franklin Lakes, NJ, USA). 

Cell proliferation assay

 A total of 2500 PDLSCs at passage 3 (P3) isolated either by enzymatic or tissue explant protocols were seeded into a 96-well plate (TPP, Trasadingen, Switzerland) and cultured in media containing either 5% PL, 10% FBS or serum-free medium (SFM). The experiment was performed daily for a total of 7 days and media were exchanged every 3 days. Proliferation rate was measured using CellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA).

Colony formation unit assay (CFU)

A total of 500 PDLSCs at P1 through 5 were obtained from enzymatic and explant methods and seeded into 20-mm cell culture dishes in either 10% FBS, 5% PL or SFM. Cells were cultured for 14 days with medium exchange every 3 days. Aggregates consisted of more than 50 cells were recognized as PDLSCs colonies. Colonies were stained using Crystal Violet dye (Sigma, St. Louis, MO, USA) followed by colonies counting under the inverted microscope (Zeiss, Cincinatti, OH, USA). 

Karyotyping

Cytogenic analysis was performed at passage 3, 5 and 7 for PDLSCs isolated by either enzymatic or explants and propagated in two different serum types. Briefly at 70% confluency, cells were treated with Colcemid 10 µg/mL (Euroclone, Italy) for a period of 4–6 h to induce cell cycle arrest at metaphase. Cells were trypsinized and centrifuged at 400 xg for 5 min. The cell pellet was resuspended in a volume less than or equal to 500 ml of alpha-MEM (Gibco, Waltham, MA, USA) and then subjected to 0.075 M KCl hypotonic solution (Euroclone, Pero, Italy), added drop by drop under continuous agitation. After 30 min of incubation at 37 °C and in the presence of KCl, cells were centrifuged at 400 xg for 10 min. Next, The cell pellet was fixed with 10 ml freshly prepared Carnoy’s solution consisting 3;1 methanol:acetic acid (Carlo Erba Regeants) for 20 min. Then, the pellet was washed twice with the same solution. After the final washing step, the pellet was resuspended in 3 ml of the same solution (carnoy’s solution), and dropped on pre-cooled glass slides. Slides were then stained using the classical GTG (G-bands after trypsin and giemsa) and the number of cells in metaphases were analysed under the light microscope at 100 X magnification and using oil immersion. A minimum of 7 metaphases were counted per sample. The analysis of these cells was performed using an imager D2 microscope (Zeiss) and images were captured using the Karyo Vision 3.1 imaging software (Borgonovo et al., 2014).

Stemness assay

Cells were passaged until P10 in either 10%  FBS or 5% PL to evaluate the self-renewal capabilities of PDLSCs. Then cells were trypsinized after reaching 80% confluency, stained with trypan blue (Gibco) and counted using a haemocytometer. For each passage, the seeding density was 10⁵ cells/25 cm² tissue culture flask. Stemness characteristics were also assessed by flow cytometry for the expression of the following stem cell markers: CD90, CD44, CD73, CD105 and the negative cocktail markers (CD34, CD45, Cd11b and HLA-DR).

Osteogenic Differentiation

For osteogenic differentiation, PDLSCs at P3 from enzymatic and explant methods were plated at 1 × 10⁵ cells per well in 6-well plates, and maintained in growth media containing either 10% FBS or 5% PL until they reached 70% confluence. After that, growth medium was replaced withosteogenic media and maintained under the same serum conditions. Osteogenic media was prepared using α-MEM supplemented with 10 mM  β-glycerophosphate (SigmaAldrich), 50 µg/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich) and 1 µM dexamethasone (Sigma-Aldrich). Medium was exchanged every 3 days for a total of 14 days after which they were processed for RNA isolation and Alizarin Red staining. 

The functionality of the cultured osteoblasts was examined by quantitative PCR (qPCR) for the following osteogenic markers: osteocalcin (OCN), alkaline phosphatase (ALP), collagen type 1, osteopontin (OPN), Osterix and Cbfa-1. Briefly, RNA was extracted from the differentiated samples, and then cDNA synthesis was performed using superscript VILO master mix (Thermo Fisher). Diluted cDNA was amplified using GoTaq® qPCR Master mix (Promega) and mixed with 300 nM of each of the forward and reverse primers (IDT, USA) (Table S1). The amplification conditions were as follow: 95 °C for 2 min as initial denaturation cycle, then 40 cycles of 95 °C for 15 s, 60 s of annealing step at 58 °C for each of OCN, OPN and ALP genes and 60 °C for each of Osterix, collagen type 1 and Cbfa-1, and 72 °C for 30 s as an extension step. Samples were run on CFX system (Bio-Rad, Hercules, CA, USA) and the relative expression levels of these genes were normalized to cDNA samples from control cells harvested at day 14 (cells cultured in osteogenic supplements-free media under the same conditions). Moreover, the relative expression levels of osteogenic genes were also normalized to cDNA samples of cells cultured under the same conditions and harvested at day1 of the differentiation process.

Statistical analysis

Data were analyzed on GraphPad Prism version 7. All assays were performed in duplicate of at least three independent experiments (n = 3). Results are expressed as Mean ± Standard Deviation. Statistical significance was determined using either a paired t-test or one-way analysis of variance (Heider et al., 2013) as appropriate. Statistical significant difference was considered when p-value < 0.05.

Morphological evaluation of PDLSCs

Enzymatic-treated and explant derived cells showed a heterogeneous morphology ranging from spindle-shaped to elongated fibroblast-like cells.  Proliferation rates were also documented and demonstrated similar potencies. PDLSCs isolated by the explant method started to expand from the attached sections on day 4, after which the cells were harvested on day 14 (Fig. 1A). The same pattern of cell growth was observed for PDLSCs isolated by enzymatic digestion method (Fig. 1B). However, the rate of cell growth was slightly higher in enzymatically-treated compared to explant derived cells, which is in line with a previously reported data (Tanaka et al., 2011).

Colony-forming capacity of PDLSCs

Colony forming cells are considered a prerequisite for osteooblast differentiation (Couble et al., 2000; Huang et al., 2006; Papaccio et al., 2006). To assess the capacity of self-renewal of the isolated PDLSCs; a colonogenic assay was performed at passages 1 through 5. As shown in Fig. 1C, enzymatic and explant PDLSCs generated the similar number of colonies. While the use of 10% FBS showed a slightly higher number of colonies compared to 5% PL. However, the difference in colony number was not statistically significant. We have also observed that colonies grown in 10 % FBS were more compact compared to 5% PL (Fig. 1D).

Surface markers analysis

Homogeneous cell population is required for successful use in therapeutic applications. To ensure the homogeneity of PDLSCs, the MSC markers expression profile was assessed by flow cytometry at P3. Flow cytometric analysis revealed that cells isolated by both methods and treated with either PL or FBS showed positive expression of MSC markers (CD44, CD105, CD90 and CD73) and negative for hematopoietic stem cell markers (CD45, CD34, CD11b and HLA-DR) as shown in Table 1 & Table S2 and Fig. 1E. These results indicate that the cells have similar expression profile to MSCs. No statistically significant difference was observed in any of the analyzed samples.

Proliferative potential of PDLSCs

To investigate the effect of the isolation method on the proliferative potential of PDLSCs, proliferation rates of the isolated PDLSCs were investigated using the CellTiter 96® Non-Radioactive Cell Proliferation Assay. Proliferation of PDLSCs isolated by enzyme-digestion method was slightly higher compared to PDLSCs obtained by the explant method (Figs. 2A & 2B). Similarly, FBS serum promoted a small increase in the proliferation rates when compared to PL in both methods. However, these differences were not statistically significant (Figs. 2A & 2B) (p value > 0.05) (Table S3).

Karyotyping analysis

To ensure that the cell culture and expansion conditions have maintained genome stability of the isolated cells, we performed a cytogenetic analysis using the classical GTG (G-bands after trypsin and giemsa) banding technique. All isolated PDLSCs exhibited 46 chromosomes including sex chromosomes, without any aneuploidy, polyploidy or chromosomal abnormalities in metaphases at passage 3, 5 and 7 (Figs. 2C–2F).

Stemness characteristics

PDLSCs need to retain their stemness characteristics, in order to maintain them in the undifferentiated state. Stemness potential was investigated by evaluating the cell proliferation using the following assays: performing cell count, assessing MSC marker expression and observing the morphological changes for up to P10. We found that PL significantly enhanced the proliferation of the derived cells in both methods and until P5 (p < 0.05) (Fig. 3A) (Table S4). Moreover, our results showed that cells isolated by both methods and treated with either PL or FBS showed positive expression of MSC markers (CD44, CD90 and CD73) and negative expression for hematopoietic stem cell markers (CD45, CD34, CD11b and HLA-DR) through passage 1–10 (Table S5). This effect was more consistent in PDLSCs isolated by explant method. After P5, a gradual loss of proliferation potential was observed till passage 10 using the two methods of isolation and both serum types. Typically, CD105 expression had dropped clearly in cells derived from explant culture compared to the enzymatically derived cells (Table S5).

Morphologically, we did not observe any difference in cell shape in any of the culture conditions from passages 3 through 5 (Fig. 3B). However, cells from both cultures changed their morphology after P7 (Fig. 3B), and a loss of homogeneity was observed in culture until P10. These changes can be attributed to cell senescence or spontaneous differentiation (Gaebel et al., 2011; Kern et al., 2006) and it might be an outcome of the loss of CD105 marker.

Osteogenic differentiation

Mineralization potential of PDLSC was also assessed to evaluate their regenerative potential. Osteogenic differentiation was induced for 14 days and calcium deposits were detected with Alizarin red staining (pH 4.2; Sigma-Aldrich). Both explant and enzymatic PDLSCs exhibited an efficient osteogenic differentiation potential by showing the same intensity of alizarin red stain compared to cells cultured under the same conditions and maintained in osteogenic supplements-free media (Blank Control) (Figs. 4A–4H).

At the gene level, cells derived from any of the two extraction methods and treated with either PL or FBS showed a significant increase in the expression of the following osteogenic genes: ALP, OPN, collagen type 1, and osterix when compared to control cells derived from day 14 (P < 0.05) (Fig. S1A-S1E) (Table S6). Whereas, the expression of Cbfa-1 has slightly increased insignificantly in differentiated cells compared to control cells, irrespective of the extraction method and the serum type used (P > 0.05) (Fig. S1F).

To determine the impact of using different extraction methods and different serum types on the efficiency of osteogenic differentiation, the expression levels of the osteogenic markers of each group were normalized to their blank controls harvested on day 14 of the osteogenic differentiation. First, the expression levels were compared between differentiated cells derived by the same extraction method and treated with different serum types. For explant method, cells treated with PL showed a statistically significant increase in the expression levels of OPN and Cbfa-1 compared to FBS (P < 0.05), Table 2, Figs. 4L & 4N. While, OCN level was higher in cells treated with FBS compared to PL (Table 2, Fig. 4I). For enzymatic method, both ALP and OCN were significantly increased in cells treated with PL compared to FBS (Table 2, Figs. 4I & 4L). After that, we performed a comparison between cells treated with the same serum type and different extraction methods. Interestingly, for both PL and FBS cells extracted by explant method showed a statistical significant increase in the expression of ALP compared to cells derived by enzymatic method (P < 0.05), (Table 3, Fig. 4L), which suggests that the combination between explant extraction method and PL is more efficient on osteogenic differentiation compared to the enzymatic derived cells, and FBS treated cells.