Enhanced human periodontal ligament stem cell viability and osteogenic differentiation on two implant materials: An experimental in vitro study

Disc preparation

Yttria stabilized tetragonal zirconia polycrystals (ZrO₂ balanced, Y₂O₃ < 5.15 mass %, HfO₃ < 3 mass%, Al₂O₃ < 0.5 mass%, SiO₂ < 0.02 mass %, Fe₂O₃ < 0.01, Na₂O < 0.04) (Bruxzir Shaded, Glidewell) and polyether ether ketone polyetheretherketone (PEEK) blocks were used. The blocks were cut into discs of 1 mm thick and 10 mm in diameter. Four experimental groups of discs were formed according to the type of material and surface treatment: experimental zirconia discs with polished surface (Z); experimental zirconia discs with a sandblasted surface (ZS); experimental PEEK discs with smooth surface (P) and experimental PEEK discs with a sandblasted surface. Using a polishing apparatus (Ecomet 3, Buehler), polished TZP samples were first ground on the two sides with diamond discs (70 m, then 45 m). They were then polished with a polishing cloth, diamond particles measuring 3 and 9 microns, and colloidal silica measuring 0.6 microns.¹³ PEEK polished samples, however, were produced by first polishing the PEEK disc with 1000 and 2000 grit silicon carbide sandpapers, then finishing with 8000 grit alumina lapping film to offer a smooth surface.¹⁴ At 50 mm perpendicular distance on all sides, particles of 110 m alumina (Korox 110, BEGO) were used to sandblast specimens of TZP and PEEK at 5 bar air pressure using a sandblaster (Basic eco, Renfert). All discs were sandblasted for 10 seconds.¹⁵ All samples underwent a 10 minutes ultrasonic cleaning procedure using acetone and distilled water before being used in cell culture experiments, followed by a half an hour UV disinfection lamp application on each side of each disc.

In-vitro stem cell isolation and characterization

Periodontal ligament (PDL) cells isolation and culture

Under the guidance of the British University in Egypt's Ethical Committee (approval # 22-003) and with patients' (n=3) full agreement and signing an informed consent, three human permanent teeth were gathered from healthy donors at the oral and maxillofacial dental department. The teeth had been extracted for orthodontic purposes. The patients were informed about the study privately and the patient’s data confidentiality was ensured. The patients’ inclusion criteria were; not suffering from any chronic diseases, and average age between 20–40 years. The patients were not further included in the study and were considered as teeth donors only. The selected teeth were free from any carious lesions.

The extracted teeth were kept in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO), which received antibiotic doses (300 U/ml penicillin and 300 mg/ml streptomycin; Sigma). Within the first 24 hours from teeth extraction, primary cell culture was performed. Sterile phosphate-buffered saline (PBS, Sigma) was used to disinfect the PDL tissues removed from healthy third molars root surfaces. PBS was then added with antibiotics at escalating doses to provide further protection. Stem cells were isolated from these PDL tissues by using the outgrowth method.¹⁶ Smaller pieces of PDL were put onto 35 mm plates (NUNC, Roskilde) with 1 mL of culture media, which was composed of Dulbecco's modified Eagle's medium/nutrient combination F-12 Ham medium (DMEM/F12, Sigma) with 10% fetal bovine serum (FBS, Gibco) added as a supplement and 1% penicillin/streptomycin. 37°C incubation was conducted with 5% CO₂ in a humid atmosphere. An inverted microscope was used to monitor cell growth and morphology. The cells were trypsinized and sub-cultured once they had reached 80% confluence. Every three days, the medium was changed. All tests were carried out on cells acquired during the fourth passage (Figure 1a,b).

Figure 1. hPLDSCs growth observation under microscope.

(a) Cell aggregates at passage 0; (b) Confluent cells at passage 4 (Mag. 40×).

Identification of isolated PDL cells as mesenchymal stem cells (MSCs)

Cell surface marker identification. The expression of specific surface markers on the extracted PDLSCs was identified by fluorescence-activated cell sorting (FACS). The cells were collected, moved, and then fixed for 15 minutes in 4% paraformaldehyde. First, 3% bovine serum albumin (BSA; Sigma-Aldrich) was used to incubate the cells before being incubated for one hour with primary antibodies (eBioscience, Thermo Fisher Scientific), produced against CD105, CD90, CD73, CD34, CD45, and HLA-DR for 1 hour. The secondary antibody was applied and left on the cells for 45 minutes at room temperature after being rinsed with wash buffer. After three rounds of washing, a flow cytometer (FACSCalibur, BD Biosciences) was used to evaluate the cells.¹⁷

Multilineage differentiation. To evaluate the in vitro potential differentiation capacity of hPDLSCs, 5×10⁴ cells/ml from the fourth passage were cultured in 24-well plate with OsteoDiff media, AdipoDiff media, and CondroDiff media (Human mesenchymal stem cell functional identification kit, R&D Systems) for three weeks to achieve osteogenic, adipogenic, and chondrogenic differentiation, respectively. Alizarin red staining (Sigma-Aldrich) was used to assess mineralization, which demonstrates osteogenesis. Oil red O solution (Sigma-Aldrich) was utilized to find lipid droplet aggregation, which demonstrates adipogenesis. Alcan blue staining (Sigma-Aldrich) was used to identify glycosaminoglycans verifying chondrogenic differentiation.

Assessment of cell viability, morphology and osteogenic differentiation capacity upon culture on disks

Cell viability. Six discs of each group (Z, ZS, P and PS) were put at the bottom of a 6-well plate. Cells were then put to the wells at a density of 30×104 cell for each well. The culturing of cells was on the discs for the following 24, 48, and 72 hours in which cellular viability was evaluated, utilizing methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay (Sigma Aldrich), relative to control cells being cultured in 6-well plates without discs. Absorbance was recorded using a benchtop microplate reader (Thermo Scientific Multiscan).¹⁸ The findings were presented as mean viability % relative to controls cultured in the absence of disks.

Cell morphology. Cells were seeded in 6-well plates on Z, ZS, P and PS discs as detailed earlier and 72 hours were spent cultivating. Subsequently, 4% glutaraldehyde was used to fix the cells for 2 hours at 4°C.¹⁹For critical point drying, samples dehydration was done using increasing ethanol concentrations from 25%,50%, 75%, 95%, to 100%, 5 minutes in each concentration. Samples were subjected to sputter-coating with gold using 15 mA for 4 min (HUMMER 8.0, ANATECH). Discs without cells and those with cultured cells were analyzed by scanning electron microscopy (SEM, Leo Supra 55).

Osteogenic differentiation assay. For osteogenic differentiation assay, cells were implanted in a 6-well plate at a density of (30 × 104 cells/well) on disks. The medium was changed the following day to osteogenic differentiation medium, which contains MEM-Alpha, 10% FBS, dexamethasone 100 nm (Sigma Aldrich, Steinheim), ascorbic acid-2-phosphate 200 uM (Sigma Aldrich), and beta glycerophosphate 10 mM (Merck, Darmstadt).²⁰ Differentiation was performed for 14 days. A positive control was achieved using cells cultivated in osteogenic media without discs, whereas a negative control used cells cultured in normal medium (DMEM). Both osteogenic induction medium and basal culture medium were altered twice per week. After 14 days of cultivation, the osteogenic distinction capacity was assessed by the calcium deposition mineralization assay using alizarin red staining and alkaline phosphatase (ALP) activity.

Alizarin red S assay. Once calcified, mineralized nodules were observed after 14 days of induction, alizarin red S staining was conducted. In more detail, the medium was aspirated and the cells were fixed with 10% formaldehyde (Sigma, v/v) at room temperature for 15 minutes. After three PBS washes to remove non-attached cells, the plates were stained for 30 minutes at room temperature with 40 mm alizarin red S (Sigma, pH 4.2). To eliminate excess stain, cells were washed 3 more times with PBS before being examined using a light microscope. Additionally, at 405 nm, the absorbance was measured after staining was solubilized in a 10% glacial acetic acid (Sigma-Aldrich) solution.²¹

Alkaline phosphatase (ALP) activity assay. ALP activity of PDLSCs on every specimen was assayed after differentiation for 14 days. Briefly, monolayers were cleaned twice with PBS and then once with 1 mL alkaline phosphatase buffer (ALPB). One mL of ALPB was added to each well and equal volumes of para-Nitrophenylphosphate (p-NPP) (Sigma Aldrich) equilibrated to 4°C were added. Immediately, 50 ul were removed and mixed with same volume of NaOH to stop the reaction. The previous step was repeated every minute for each well till 10 minutes had passed. The rate of accumulation of p-nitrophenolate was plotted for each well and the initial rate of the reaction, a way to express the rate of the reaction, was calculated by obtaining the gradient at the linear phase for each group. Monolayers were then cleaned in PBS and kept at -20°C for total protein quantification.²² Total protein amounts were detected utilizing enhanced BCA protein assay kit (Pierce) based on the manufacturer’s procedures.²³ The absorbance at 405 nm was detected by spectrophotometry. The initial rate of the reaction in each group (slope) was divided by protein concentrations (mg).

Statistical analysis

Each in-vitro assay was carried out in triplicate and analyzed across three distinct studies. Graph-Pad Prism v8.1.0 (RRID:SCR 002798) was used to conduct ANOVA test, followed by a pair-wise Tukey's post hoc test, after validating the homogeneity of variance and normal distribution of the data.

PLDSCs identification

According to flow cytometry findings, isolated PLDSCs were CD73 (98.76%), CD90 (95.67%), and CD105 (95.52%) positive; and less than 5% for CD34 (4.44%), CD45 (4.48%), and HLA-DR (2.66%) (Figure 2a). The rates are provided in respect to the presence of mesenchymal stem cell surface markers. Additionally, hPDLSCs were found to be capable of differentiating into osteoblasts, chondrocytes, and adipocytes (Figure 2b). PLDSCs differentiated into osteoblasts showed positive red staining of the mineralized matrix using alizarin red S stain, cells differentiated into chondrocytes showed positive staining of the produced glycosaminoglycans via alcian blue stain and cells differentiated into adipocytes showed positive staining of the oil droplets using oil red staining.

Figure 2. PLDSCs identification (a) Flowcytometry Figures (b) In vitro multilineage cell differentiation (Mag. 100×).

Cytotoxicity assay

An MTT assay was utilized to determine the viability of the hPLDSCs (Figure 3). Data are presented after 24,48, and 72 hours of exposure of the PLDSCs to discs and were normalized to the control (cells + medium). Polished PEEK surface discs showed a notably reduced cell viability % in comparison to the three other groups at 24,48 and 72 hours (p<0.05). The sandblasted roughened zirconia and PEEK discs showed increased cell viability % throughout the whole experiment, the roughened PEEK surface possessed significant higher cell viability % compared to the same respective group of smooth polished surface (p<0.001), and the roughened Zirconia discs also showed higher cell viability % compared to the same respective group of smooth polished surface (p<0.05). However, the percentage of cell viability was not significantly different between the roughened zirconia and PEEK discs.

Figure 3. MTT assay.

The data are presented as absorbance values (570 nm) at 24, 48, and 72 h of the PLDSCs being exposed to discs. *p significant difference of the respective group in comparison to the Z group, ^#p significant difference of the respective group in comparison to the P group, p values <0.05 are statistically significant. For each material's surface properties, each experimental condition was run in triplicate.

Cell adhesion and morphology

The discs’ surface microstructure and stem cell morphology are shown in Figure 4. Cells with extending cytoplasmic processes were frequent and well-observed in smooth polished zirconia discs while no well-defined cells were seen attached to the polished PEEK discs. Cell adhesion and structure of hPLDSCs on roughened sandblasted specimen surfaces revealed the presence of numerous well-attached-type cells with extending stretched cytoplasmic processes.

Figure 4. Representative SEM micrographs illustrate (a) polished smooth zirconia surface, (b) cell adhesion on polished smooth zirconia surface, (c) sandblasted roughened zirconia surface, (d) cell adhesion on sandblasted roughened zirconia surface, (e) polished smooth PEEK surface, (f) cell adhesion on polished smooth PEEK surface, (g) sandblasted roughened PEEK surface, (h) cell adhesion on sandblasted roughened PEEK surface, (i) cell adhesion on sandblasted roughened PEEK higher resolution.

Mineralization assay calcium deposition

On day 14, extracellular matrix rich in calcium was assessed as a byproduct of the osteogenic differentiation process when stained with alizarin red S (Figure 5). There were notable variations in between the groups examined; the negative control (cells+normal medium) displayed fewest mineralized nodules amounts; the positive control (osteogenic medium only) exhibited higher amount of calcium deposition (p<0.001) than polished zirconia and PEEK groups (Figure 5). When compared to the other experimental groups, roughened sandblasted zirconia and PEEK groups possessed greatest grades of mineralized nodules (p<0.001). Average mean values of absorbance in the control, Osteo, Z, P, ZS, and PS groups are 0.048±0.01, 0.24±0.01, 0.1784±0.01, 0.11±0.02, 0.33±0.01, 0.32±0.01, respectively.

Figure 5. Osteogenic differentiation and alizarin assay: On day 14, as a byproduct of the differentiation of osteoblasts, calcium deposition was examined using alizarin red S staining.

(Mag. 40×). (a) Control; (b) Osteogenic medium; (c) Smooth zirconia surface; (d) Smooth PEEK surface; (e) Roughened zirconia surface; (f) Roughened PEEK surface. Every experimental condition was carried out in triplicate for every material surface characteristics. (g) Alizarin red staining data from three independent experiments were statistically analyzed and given as mean standard deviation, *p significant difference of the respective group compared to the control, ^#p significant difference of the respective group in comparison to the osteo group. ^$p significant difference of the respective group in comparison to the Z group. p values <0.05 is statistically significant.

ALP (Alkaline phosphatase) assay results

The ALP assay kinetic profile demonstrated the aggregation of the yellow p-nitrophenolate (p-NP) as a growing product among the various groups (shown in Figure 6a). The control group possessed the lowest rate of accumulation, the polished zirconia and PEEK showed lower rate of p-NP accumulation than the roughened sandblasted disks of same respective groups. Statistical analysis was done by calculating the slope of each curve and divided by the total amount of protein in each well. The results showed that the ALP activity at day 14 in cells cultured on the polished zirconia and PEEK discs was significantly lower than the positive control (p<0.05); polished PEEK discs possessed the lowest ALP activity compared to the polished zirconia discs and positive control (p<0.001). Roughened sandblasted Zirconia and PEEK discs indicated most ALP activity in comparison to the polished discs (p<0.001) (Figure 6b). Regarding ALP activity, no discernible change was found in cells cultured on the roughened sandblasted zirconia discs, PEEK discs and the positive control group, suggesting that roughened sandblasted surfaces can stimulate the osteogenic potential of PDLSCs. Mean values of the rate of ALP reaction (slope) of the control, Osteo, Z, P, ZS and PS groups are 0.0038±0.0002, 0.0125±0.001, 0.0107±0.001, 0.009±0.004, 0.0131±0.001, 0.0135±0.0003, respectively.

Figure 6. ALP (Alkaline phosphatase) activity after 14 days of osteogenic induction.

a) An illustration of an ALP assay's kinetic curve showing how different groups over time accumulated the yellow p-nitrophenolate product. b) The initial rate of the reaction in each group by calculating the slope, values were scaled back to the quantity of total protein. The values revealed as mean±SD of three independent experiments, *p significant difference of the respective group in comparison to the control (cells in normal medium), ^#p significant difference of the respective group in comparison to the osteo group (osteogenic medium), ^$p significant difference of the respective group compared to the Z group. p values <0.05 is statistically significant.