Comparing the Adhesion Capability of Periodontal Ligament Fibroblast Cells to Nano-hydroxyapatite Silicate-Based Cement and Silicate-Based Cement Alone

Hydraulic silicate cements have the ability of apatite core formation and can stimulate tissue repair, osteogenesis, and cementogenesis(1–3). Silicate-based cement alone enjoys good handling, which is a critical feature as a bone substitute material (4). Another material with successful results in implant dentistry and regenerative processes is Hydroxyapatite (HA). HA, in the form of powder, has low clinical handling capability; however, due to its excellent compatibility with hard and soft tissue, it is an ideal material for implant dentistry. Recently, many studies have been carried out on the form of Nano hydroxyapatite (4, 5–10). A Nano-sized hydroxyapatite (less than 100 nanometers in size) is ideal because it can stimulate inorganic phase in the bone (11, 12); on the other hand, HA Nanoparticles with an increased ratio of surface to volume may cause an increase in cell adhesion, proliferation, and osteoblast adhesion (8, 13). In a recent study, the effect of silicate-based cement and its combination with Nano-HA has been evaluated on implant primary stability and it was concluded that both cements could equally increase primary stability (4).

The aim of this study was to compare the adhesion capability of periodontal ligament fibroblast cells to the Nanohydroxyapatite silicate-based cement and silicate-based cement alone in vitro. In addition, the cellular proliferation and attachment of PDLFCs to test materials were examined to observe the physical properties of the cells following adding Nanohydroxyapatite particles.

Materials and Methods

Periodontal ligament fibroblast cells culture

                Ten third molar teeth (extracted from healthy subjects due to non-pathological problems) were collected and placed in a basic medium [α-MEM (Minimum Essential Medium α) supplemented with 10% FBS (fetal bovine serum)]. The samples were transferred to the laboratory at 4 ° C. The PDL tissue separation was performed under sterile conditions, within a biohazard laminar flow hood for each tooth. Tooth surfaces were first cleaned with 70% ethanol. A scalpel blade was used to remove PDL from the root surface. Tissue was cut into small pieces (smaller than 0.5 mm) and cultured in cell culture plates in DMEM containing 10% fetal bovine serum and 0.5% antibiotics (diluted from a stock solution containing 5000 U/ml penicillin and 5000 U/ml streptomycin) at 37º C in an atmosphere of 100% humidity and 5% CO2. The cells that grew were detached from the culture flask using 0.05% (w/v) trypsin and 0.05 mM (w/v) EDTA and transferred to 24-well-plates where they were subcultured at a density of 10000 cells/well.

2.2. Cell suspension and Counting

                The 0.5 ml of cell suspension was taken using a sterile pipette and placed in an Eppendorf tube. 100 µL of cells were transferred to another Eppendorf tube and 400 µL of Trypan Blue 0.4% was added and slowly mixed.

Pipettes were used to obtain 100 μL of trypan blue treated cell suspension and applied to a homocytometer. The microscope was used to focus on homocytometer grid lines with a 10-fold objective. Live cells (Live cells do not stain with Trypan Blue) were counted in a set of 16 squares.

                For determining the number of cells/ml and the total number of cells the following formula was used:

Total # cells = cells/ml vol. of original cell suspension

To calculate the percentage of viable cells the following formula was used:

Experimental design

In this study we had 3 groups: Group 1 Silicate-Based Cement alone (APC): 90% Portland cement type 1(Fars Cement Co, Iran) with 10% Calcium Chloride (Kimia Material Co, Iran) additive - using a volumetric spoon. Group 2 Nanohydroxyapatite silicate-based cement (APC +Nano-HA): Based on previous experience (4), the ratio of the two materials was set to 25% APC in combination with 75% Nanohydroxyapatite (PardisPajouheshFanavaran Yazd, Iran) - using the volumetric spoon. Group 3 Control: PDLFCsseeding on a culture plate (as control). Each test group materials mixed with distilled water for setting reaction. APC was mixed with water at a 2: 1 ratio; APC +Nano-HAwas mixed with water at a 3: 1 ratio. Scanning electron microscopy by a TESCAN scanning electron microscope (VEGA 3 – TESCAN, Czech Republic) at ×5k magnification was used to evaluate the surface morphology. Zeiss EM10C transmission electron microscope (TEM) at 80 kV (Zeiss, Germany) was performed to obtain the morphology and size of the nanoparticles.

The mixed test materials placed into 48-well plates (8-well plates for each material for 3 timing periods of 24h, 72h and 1 week). Each experiment was done triplicate.

MTT assay was used to evaluate the survival and proliferation of fibroblasts on days 24, 72 and 1 week after cell culturing. A fresh culture medium with 10% MTT was added to each well, and the plates at 37 ° C Incubated for 4 hours. The culture medium of each well was extracted and replaced with dimethyl sulfoxide solvent. Then, 100 μl of each purple solution was transferred to a 96-well Elisa reader plate. The absorbance of the dye was measured by Elisa reader (POLARstar Omega, Germany) at 570 and 630 nm. Optical density (OD, absorbance) of wells was used to calculate the percentage of survival of cells in each experimental group relative to the control group.                    

                      Cell Survival = OD Test / OD control * 100

The morphology of PDLFC in two scaffolds was examined by scanning electron microscopy (S-750, Hitachi, and Tokyo, Japan), then PDLFCs were seeded in each scaffold in 24 well plastic culture plates. Forty-eight hours after initial seeding, the culture medium was discarded and cells were washed slowly with a solution containing phosphate three times. Cells were fixed on scaffolds with 2.5% glutaraldehyde for 12 hours. The fixative removed and the scaffolds were carefully washed with phosphate-buffered solution.

Then the scaffolds were exposed for 15 minutes to sequential dehydration with a series of ethanol (50%, 70%, 80%, 90% and 95%). The isoamyl acetate replaced and the scaffolds were allowed to dry at a typical critical point, then coated with gold using an ion-sputtering coater (IB-5, Eiko, Japan) and subjected to a scanning electron microscope for evaluation of cell attachment and morphology.

Data analysis

In the MTT analysis, mean values and standard deviation were calculated for each group.Differences for each material between the three-time periods tested according to the analysis of variance (ANOVA) and one-way analysis of variance were also performed for the material with significant difference by ANOVA. Differences between the two substances for each time period were assessed using Student-t test.The significance level was considered to be 0.05.

This study was carried out in accordance with the guidelines of the Declaration of Helsinki as revised in Edinburgh (1975). The study protocol was approved by the vice chancellery for research affairs of Shiraz University of Medical Sciences, Shiraz, Iran (code: 95-01-03-11625 and 10018). A written and verbally informed consent was obtained from all participants.3. Results

SEM for Surface characteristics

APC + nanoHA surfaces showed irregular mixed surface features including round and/or sharp and cuboidal crystals. (Figure 1)

Imaging Transmission Electron Microscope (TEM) of hydroxyapatite nanoparticles by means of fest electron presented in figure 2.The size of nanoparticles is about 20-25 nanometers. 

Result

Table I shows MTT data analysis and statistics of two different substrate (APC&APC+nano-HA) for 24, 72 h, and 1 week. In terms of cell population, data analysis of MTT assay reflects no statistically significant difference between APC &APC+nano-HA groups at 24 h and 1 week (P = 0.511 & 0.162 respectively). Although there is a significant difference between APC &APC+nano-HA groups at 72 h for the benefit of the APC group (p=0.016).

Fig. 3 shows the cell number on the APC substrates determined by the MTT assay 24 , 72 h, and 1 w after seeding. The cell population after 24 h reflects initial adhesion on the APC substrates, and the great increase in the population after 72 h indicates subsequent proliferation of the cells while there is a decline in cell population after 1week to the level of the initial adhesion at 24 h. MTT data analysis shows statistically significant differences between three times .Table 1 and II. (P = 0.001)

On the other hand, the cell population of APC+nano- HA substrates After 24 , 72 h and 1 week of seeding, reflects initial adhesion on the substrates with no statistically significant difference between three times .Table I. (P = 0.388). 

Figure 3. The number of cells on each APC & APC+ Nano-HA pellet 24 h, 72 h and 1week after seeding determined by MTT assay.

Table I. MTT data analysis and statistics of two different substrates (APC&APC +Nano-HA) for 24, 72 h and 1 week

Table II. Multiple comparison analysis of different time periods of seeding

Scanning Electron Microscopy (SEM) for Cellular analysis

Fibroblast cells showed good adaptation and also showed spreading cytoplasmic extensions which are essential for attachment and the proliferation of cells. A variety of cellular forms was observed. (Figure 4) The variety of cellular morphologies indicating the movement and proliferation of fibroblasts in the test materials.

Figure 3. The number of cells on each APC & APC+ Nano-HA pellet 24 h, 72 h and 1week after seeding determined by MTT assay.