Ethical approval
This study was approved by the research ethical committee at Faculty of Dentistry, Ain Shams University, Egypt (Approval No: FDASU-RecID051907 in 17/7/2019). Moreover, all methods were carried out in accordance with relevant guidelines and regulations. Informed consent was not obtained because no humans and/or human tissue samples were involved in this study.
Sample size calculation and classification
Based upon the previous study of Abu Zeid et al., the effect size (d) was 1.118 with success of MTA = 69.2%. Using alpha (α) level of (5%) and Beta (β) level of (20%) i.e. power = 80%; the minimum estimated sample size was 28 for tested materials and 14 samples for negative control. Sample size calculation was performed using G*Power Version 3.1.9.225.
The investigated materials were methodically crafted into disc-shaped structures (N=28 discs), with each disc acting as a representative sample. The samples were divided into two groups (14 samples each) based on the substance used: group A included MTA Angelus (ANG), and group B contained MTA Matreva. Group C (14 samples) represented the negative control (NC), which contained BM-MSCs without testing material.
Preparation of the tested materials
MTA Matreva (Matreva BioMTA®, Matreva dental labs co., Egypt) and MTA Angelus (MTA Angelus®, Angelus, Londrina, Brazil) powder-to-liquid ratios of 3:1 were combined under sterile circumstances and pressed onto 96-well plates (4 mm radius and 2 mm height) to form small discs of each material. The materials were incubated with damp gauze until hardened, then dried in a laminar hood for 24 hours before cell seeding. This was done a day before the experiment.
Isolation and culture of bone marrow mesenchymal stem cells
Stem cells were isolated from the bone marrow of two 12-week-old albino rats, tibiae and femurs weighing 200 mg each. BMMSCs showed positive CD29 and CD90 levels of 98.5% and 98.6%, respectively. Only 0.7% of hematopoietic cells expressed CD45, demonstrating that the isolated BM-MSCs were pure mesenchymal stem cells. The two albino rat samples were collected from Faculty of Medicine's animal housing facilities, Ain Shams University, Egypt.
Each rat's femurs and tibiae were immediately dissected, the connected muscles were removed, and the bones were transferred to the laboratory within a 30-minute period after being immersed in an ice-cold solution containing minimum essential medium (MEM) supplemented with 100 U/mL penicillin and 100 µg/ml streptomycin in a 15-ml Falcon tube. All treatments were performed at Faculty of Dentistry's Central Laboratory for Stem Cells and Biomaterials Applied Research, Ain Shams University. When the bones arrived at the laboratory, the bone epiphyses were removed, pulverized using a homogenizer, and incubated in 1 mL of ice cold solution comprising MEM (Gibco, Thermosientific, Germany) in sterile 15 mL Falcon tubes with 0.2% collagenase type I and 0.2% dispase (Sigma Aldrich, USA) plus 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Thermosientific, Germany) for 30 minutes at 25°C.
To eliminate bone debris, gently flip the tube several times and filter the mixture through a 100 µm cell filter. The cell suspension was centrifuged for 10 minutes at 400 ×g at 25°C in a room, followed by three rinses with cold phosphate buffer saline (PBS, Gibco, Thermosientific, Germany). The supernatant was then collected and disposed away. The cells were then suspended in DMEM with 10% fetal bovine serum (FBS, Gibco, Thermosientific, Germany) and antibiotics (penicillin/streptomycin) and incubated at 37°C for 24 hours with 5% CO2 and 100% humidity.
The following day, 1x104 BMMSCs were seeded in 200 µL of mesenchymal stem cell (MSC) osteogenic differentiation medium (OM, Gibco, Thermosientific, Germany), which included optimized MSC osteogenic differentiation basal medium, mesenchymal stem cell-qualified fetal bovine serum, penicillin, streptomycin, glutamine, ascorbate, β-glycerophosphate, and dexamethasone. The culture plates were incubated at 37°C in a 5% CO2 environment for the times specified. BM-MSCs grown on OM without MTA material served as negative controls.
Assessment of cell viability and cytotoxicity
The MTT was used to assess cell viability after 3, 7, and 14 days. Cell proliferation was evaluated with the Vybrant® MTT Cell Proliferation Assay Kit (Cat no: M6494, Thermo Fisher, Germany). After the incubation time, 100 µL of media was replaced with fresh media. Next, 20 µL of MTT solution (1 mg/mL) was applied to each well. At each time point, a cell proliferation test was performed. The experimental design was intended to analyze the proliferative capability of BMMSCs on the studied MTA materials at three time points: 3, 7, and 14 days. The plates were incubated for four hours at 37°C with 5% CO2. After removing the MTT solution, 100 μL of sodium dodecyl sulfate with hydrochloric acid (SDS-HCL) was applied to each well. The optical density (OD) of 570 nm was measured to detect cell viability.
Assessment of osteogenic potential
Assessment of osteogenic potential through evaluation of calcium deposition by Alizarin red staining (ARS)
The tested materials were used to direct the osteogenic differentiation of BM-MSCs into osteoblasts. The cells were grown for fourteen days. Following the incubation period, calcium deposition in differentiated osteoblasts was examined by ARS, which evaluated calcium buildup in mature osteoblasts.
Cells were planted onto MTA Matreva and MTA Angelus discs in 96-well plates and cultured for 14 days at 37°C in a 5% CO2 incubator. After 14 days, the growth media were removed and the cells were washed three times with PBS. After 15 minutes of fixation with 4% formaldehyde at room temperature, the cells were washed three times with deionized water (diH2O). A single 40 mM mL of ARS was applied and shaken at room temperature for 30 minutes. After five washes with diH2O, the cells were analyzed via an LX400 fluorescence microscope (Labomed, Los Angeles, California, United States).
The assessments were repeated three times to assure accuracy. For each group, the H-score method was utilized to first recognize individual cells and their subcellular compartments, followed by the relative expression of ARS [area fraction] (Loba Chemie Pvt. Ltd., Mumbai, Maharashtra 400005, India) to rate the staining intensity as high (3+), medium (2+), low (1+), or no staining (0). The H-score was determined by dividing the sum of area fraction intensity by the total number of detected cells26.
Assessment of osteogenic potential by RANKL protein expression
The expression of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) protein in BM-MSCs seeded on the two MTA materials for 14 days was evaluated. The tested materials were used to direct the osteogenic differentiation of BM-MSCs into osteoblasts. The cells were grown for fourteen days. After incubation, cells were collected using 0.25% trypsin, washed three times with PBS, and centrifuged at 1000×g for 10 minutes. The resultant cell pellet was then resuspended in culture medium, with the cell count set to1x106 cells/ml. RANKL protein expression was evaluated in collected cell homogenates using an enzyme-linked immunosorbent assay (ELISA) kit (Fine test, Wuhan, China). The BM-MSCs in the two separate groups were seeded for 14 days, as per the research design. The BM-MSCs were quickly immunostained with a rabbit anti-RANKL antibody (Invitrogen; ThermoFisher Scientific, Hilden; Germany) and examined under a microscope. The H-score median value was calculated27.
Assessment of osteogenic potential through gene expression
The tested MTA materials were used to direct differentiation of BM-MSCs into osteoblasts. The cells were grown for fourteen days. Following the incubation time, total mRNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions.
Firstly, complementary DNA (cDNA) was generated by reverse transcription with a QuantiTect II RT Kit (Qiagen, Hilden, Germany). Secondly, gene expression analysis involved quantifying the gene expression levels of the transforming growth factor β subunit (TGF-β), tumor necrosis factor α subunit (TNF-α), and interleukin 1β (IL-1β). The mRNA was subjected to gene expression amplification utilizing the QuantiTect primer set, namely, the HsTNF-α, TGF-β, and IL-1β primer sets (cat no: 249900), together with the QuantiTect SYBR Green PCR Kit (Cat no: 204141, Qiagen, Germany). The ACTB_1_SG QuantiTect Primer Assay Kit (catalog number 249900) was used as the reference gene. A 5 plex Rotor-Gene PCR Analyzer (Qiagen, Germany) was used to assess all the samples. The 2∆∆Ct technique was used to analyze gene expression levels, with ACTB serving as an endogenous reference control for normalization28.
Statistical analysis
The data were shown as mean, standard deviation, median, and range. The Kruskal Wallis test was used to compare between groups, followed by the Mann Whitney U test for pairwise comparisons. Significance level for statistical tests was set at P < 0.05. Statistical analysis was performed using SPSS software (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp)