Preparation of Titanium Discs:
Commercially grade 5 titanium was processed into discs with 5 mm diameter and 3 mm thickness using the same cutting technique for all disks with a wirecut machine (ROBOFIL 310, Charmilles technologies). Disc surfaces were mechanically polished.
In all experiments, fabricated Ti substrata were cleaned in an ultrasonic device, washed three times in distilled water, left in 70% ethanol for 10min, and washed another five times before plating the cells.
Blood Collection
Blood samples were obtained from a healthy 28 year-old, nonsmoker Iranian woman, after obtaining her written informed consent. This study was approved by the Ethics Committee of Tabriz University of Medical Sciences.
Preparation of CGF
After obtaining a signed informed consent from three systematically healthy patient with no history of any drug use, peripheral venus blood from Brachial vein was collected and promptly centrifuged by a Medifuge centrifugation framework (Silfradent, Italy). CGF clots confined from the three blood samples were blended, set on sterile gauze to dispose of abundance serum and exchanged to fereezing tubes. CGF clots were minced, homogenized, put away at -80°C for 1 h, and after that centrifuged at 3000 rpm for 10 min at room temperature. The superior liquid (CGFe) were sifted by a 0.22 μm sterile syringe filter (Memberan, CA) and after that put away at -80°C till use. The ultimate CGFe concentrations (5%, 10%, 20%, 40% and 80%) were diluted by addingculture medium containing 10% fetal bovine serum (FBS) (Biochrom, Germany) to the CGFe.
Cell Culture
A HGF cell line (HuGu, IBRC C10459) was obtained from Iranian Biological Resource Center (IBRC, Tehran, Iran).
Determine the proper concentration of CGF
The HGF cells were seeded on a 96-well plate (SPL, South Korea) at a density of 5000 cells per well and were cultured in complete medium consisting of Dulbecco’s modified Eagle’s medium (DMEM)(Gibco, USA) + 10% fetal bovine serum (FBS). After 24 hours, complete medium was exchanged and different concentrations of CGF was added (5%, 10%, 20%, 40%, 80%). Normal medium was used as the control group. After 24 hours WST-8 kit (Orangu, Cell Guidance Systems, UK) was used to analyze the cell numbers. The optical density values were measured using a microplate reader (Bio-Tek, USA) at 450 nm. The results from different groups were compared.
Cell Viability
The cells after passage four were seeded into 48 culture dishes (SPL, South Korea) at density of 14,000 cells per dish in complete medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) + 10% fetal bovine serum (FBS). After 24 hours, complete medium was exchanged.
Eighteen well were set and incubated as follows: three cultures were supplemented with 10% FBS. (Negative control group), three cultures received 10% FBS+40% CGF (positive control group), three received Titanium Discs +10%FBS (test group 1) and three received Titanium Discs +10%FBS + 40% CGF (test group 2).
After a culture period of 24 hours, HGF viability were evaluated with WST-8.
10 μl of WST-8 solution was added to each wells and incubated for one hour; Then, Orangsolution was transferred into wells of a 96-well plate.
The optical density values were evaluated with a microplate reader use at 450 nm.
Statistical analysis
The data are presented as mean and SD values. Differences between the control and test groups over time were tested using Mann–Whitney Utest. The level of statistical significance was set at p<0.05.